Optimized methods for differentiation of cells into cells with hepatocyte progenitor phenotypes, cells produced by the methods, and methods of using the cells

ABSTRACT

The invention is directed to methods for culturing cells so that the cells are induced to differentiate into cells that express hepatocyte phenotypes and hepatocyte progenitor phenotypes. More particularly, the invention relates to methods for culturing cells so that the cells are induced to differentiate into cells that express a definitive endodermal phenotype, a liver-committed endodermal phenotype, a hepatoblast phenotype, and hepatocyte phenotype. The invention is also directed to cells produced by the methods of the invention. The cells are useful, among other things, for treatment of liver deficiency, liver metabolism studies, and liver toxicity studies.

FIELD OF THE INVENTION

The invention is directed to methods for culturing cells so that thecells are induced to differentiate into cells that express a hepatocytephenotype and/or hepatocyte progenitor phenotype. More particularly, theinvention relates to methods for culturing cells so that the cells areinduced to differentiate into cells that express a definitive endodermalphenotype, a liver-committed endodermal phenotype, a hepatoblastphenotype, and hepatocyte phenotype. The invention is also directed tocells produced by the methods of the invention. The cells are useful,among other things, for treatment of liver deficiency, liver metabolismstudies, and liver toxicity studies.

BACKGROUND OF THE INVENTION

Liver failure remains a devastating syndrome resulting from the loss ofhepatic cell mass below a critical level. Although the prognosis ofpatients is greatly improved by orthotopic liver transplantation,treatment is limited by worldwide shortages of donor organs. In order toovercome these problems, alternative approaches, such as bio-artificialliver devices, albumin dialysis and cellular based therapy are beingevaluated. In recent years, the feasibility to repopulate the liver withdifferent cell types, such as mature and fetal hepatocytes, embryonicstem cells, intrahepatic progenitor cells and bone marrow derived cells,have been assessed in various animal models of liver disease.

Liver Development

Mouse embryonic and fetal liver development can be divided intodifferent consecutive steps. During gastrulation (ED6-ED6.5), futuredefinitive endodermal and mesodermal cells migrate through the primitivestreak, located at the prospective posterior and proximal-lateral poleof the embryo. First, anterior endodermal cells ingress the primitivestreak, migrate towards the distal tip of the epiblast cup and displacethe visceral endoderm. The mesoderm migrates between the epiblast andendoderm. Definitive endoderm is characterized by the transientexpression of primitive streak markers (LHX1, MIXL1, WNT3, LHX1,brachyury) and CXCR4, Sox17, HNF3b, Goosecoid and E-Cadherin. Incontrast, primitive endoderm (visceral and parietal endoderm), whichgives rise to the yolk sac, expresses Sox17, Sox7, and HNF3B. Aftergastrulation, embryonic progenitors of the digestive and respiratoryorgans initially exist in a single cell thick, epithelial sheet ofendoderm that lines the ventral surface of the embryo. Then, theendoderm folds into a gut tube to form the foregut, midgut and hindgutendoderm. At ED8.25, ventral foregut is guided towards a hepatic fateunder the influence of cytokines secreted by the adjacent cardiacmesoderm (aFGF-bFGF) and septum transversum mesenchyme (BMPs). Afterthis specification (ED0.5-ED10), the resident cells of the primitiveliver bud, consisting of bipotential hepatoblasts, undergo balancedevents including proliferation, apoptosis, and differentiation toeventually constitute a functioning organ. This further maturationoccurs through fibroblast growth factors (aFGF-FGF4-FGF8), Wntsignaling, factors secreted by the invading endothelial cells, thetransiently (ED10) present hematopoietic cells in the fetal liver(Oncostatin M) and from the surrounding non-parenchymal cells (HGF). AtED14, bipotential hepatoblast become either fully mature hepatocytes orcholangiocytes. This determination depends upon the TGBβ/Activin andNotch2/Jagged1 signaling pathway.

SUMMARY OF THE INVENTION

The invention is based on methods developed by the inventors to producea renewable source of hepatocytes in vitro. Cell culture conditions weredeveloped in view of gene expression during embryonic liver development.The inventors developed specific cell culture conditions to successfullyproduce cells that express phenotypes of hepatocytes and endodermalprogenitors. Numbered statements of the invention are as follows.

1. A method for inducing cells to differentiate into cells with ahepatocyte phenotype, comprising:

-   -   (a) culturing cells with about 5 ng/ml to about 500 ng/ml Wnt3a        and about 10 ng/ml to about 1,000 ng/ml ActivinA;    -   (b) then culturing the cells of step (a) with about 1 ng/ml to        about 100 ng/ml bFGF and about 5 ng/ml to about 500 ng/ml BMP4;    -   (c) then culturing the cells of step (b) with about 5 ng/ml to        about 500 ng/ml aFGF, about 1 ng/ml to about 100 ng/ml FGF4 and        about 2.5 ng/ml to about 250 ng/ml FGF8b; and    -   (d) then culturing the cells of step (c) with about 2 ng/ml to        about 200 ng/ml HGF and about 10 ng/ml to about 1,000 ng/ml        Follistatin.

2. The method of statement 1, wherein the cells are cultured in step (a)with about 50 ng/ml Wnt3a and about 100 ng/ml ActivinA.

3. The method of statement 1, wherein the cells are cultured in step (b)with about 10 ng/ml bFGF and about 50 ng/ml BMP4.

4. The method of statement 1, wherein the cells are cultured in step (c)with about 50 ng/ml aFGF, about 10 ng/ml FGF4 and about 25 ng/ml FGF8b.

5. The method of statement 1, wherein the cells are cultured in step (d)with about 20 ng/ml HGF and about 100 ng/ml Follistatin.

6. A method for inducing cells to differentiate into cells with ahepatocyte phenotype, comprising:

-   -   (a) culturing the cells with about 50 ng/ml Wnt3a and about 100        ng/ml ActivinA;    -   (b) then culturing the cells of step (a) with about 10 ng/ml        bFGF and about 50 ng/ml BMP4;    -   (c) then culturing the cells of step (b) with about 50 ng/ml        aFGF, about 10 ng/ml FGF4 and about 25 ng/ml FGF8b; and    -   (d) then culturing the cells of step (c) with about 20 ng/ml HGF        and about 100 ng/ml Follistatin.

7. Cells produced according to any of the methods described herein.

8. A method for inducing cells that express a primitive endodermalphenotype into cells that express a definitive endodermal phenotype,comprising:

-   -   (a) culturing cells that express a primitive endodermal        phenotype with about 5 ng/ml to about 500 ng/ml Wnt3a and about        10 ng/ml to about 1,000 ng/ml ActivinA.

9. The method of statement 8, wherein the cells are cultured with about50 ng/ml Wnt3a and about 100 ng/ml ActivinA.

10. A method for inducing cells that express a primitive endodermalphenotype into cells that express a definitive endodermal phenotype andthen into cells that express a liver-committed endodermal phenotype,comprising:

-   -   (a) culturing cells that express a primitive endodermal        phenotype with about 5 ng/ml to about 500 ng/ml Wnt3a and about        10 ng/ml to about 1,000 ng/ml ActivinA; and    -   (b) then culturing the cells of step (a) with about 1 ng/ml to        about 100 ng/ml bFGF and about 5 ng/ml to about 500 ng/ml BMP4.

11. The method of statement 10, wherein the cells in step (a) arecultured with about 50 ng/ml Wnt3a and about 100 ng/ml ActivinA, and thecells in step (b) are cultured with about 10 ng/ml bFGF and about 50ng/ml BMP4.

12. A method for inducing cells that express a primitive endodermalphenotype into cells that express a definitive endodermal phenotype,then into cells that express a liver-committed phenotype and then intocells that express a hepatoblast phenotype, comprising:

-   -   (a) culturing cells that express a primitive endodermal        phenotype with about 5 ng/ml to about 500 ng/ml Wnt3a and about        10 ng/ml to about 1,000 ng/ml ActivinA;    -   (b) then culturing the cells of step (a) with about 1 ng/ml to        about 100 ng/ml bFGF and about 5 ng/ml to about 500 ng/ml BMP4;        and    -   (c) then culturing the cells of step (b) with about 5 ng/ml to        about 500 ng/ml aFGF, about 1 ng/ml to about 100 ng/ml FGF4 and        about 2.5 ng/ml to about 250 ng/ml FGF8b.

13. The method of statement 12, wherein the cells in step (a) arecultured with about 50 ng/ml Wnt3a and about 100 ng/ml ActivinA, thecells in step (b) are cultured with about 10 ng/ml bFGF and about 50ng/ml BMP4, and the cells in step (c) are cultured with about 50 ng/mlaFGF, about 10 ng/ml FGF4 and about 25 ng/ml FGF8b.

14. A method for inducing cells that express a primitive endodermalphenotype into cells that express a definitive endodermal phenotype andthen into cells that express a liver-committed phenotype and then intocells that express a hepatoblast phenotype and then into cells thatexpress a hepatocyte phenotype, comprising:

-   -   (a) culturing cells that express a primitive endodermal        phenotype with about 5 ng/ml to about 500 ng/ml Wnt3a and about        10 ng/ml to about 1,000 ng/ml ActivinA;    -   (b) then culturing the cells of step (a) with about 1 ng/ml to        about 100 ng/ml bFGF and about 5 ng/ml to about 500 ng/ml BMP4;    -   (c) then culturing the cells of step (b) with about 5 ng/ml to        about 500 ng/ml aFGF, about 1 ng/ml to about 100 ng/ml FGF4 and        about 2.5 ng/ml to about 250 ng/ml FGF8b; and    -   (d) then culturing the cells of step (c) with about 2 ng/ml to        about 200 ng/ml HGF and about 10 ng/ml to about 1,000 ng/ml        Follistatin.

15. The method of statement 14, wherein the cells in step (a) arecultured with about 50 ng/ml Wnt3a and about 100 ng/ml ActivinA, thecells in step (b) are cultured with about 10 ng/ml bFGF and about 50ng/ml BMP4, the cells in step (c) are cultured with about 50 ng/ml aFGF,about 10 ng/ml FGF4 and about 25 ng/ml FGF8b, and the cells of step (d)are cultured with about 20 ng/ml HGF and about 100 ng/ml Follistatin.

16. A method for inducing cells that express a definitive endodermalphenotype into cells that express a liver-committed phenotype,comprising

-   -   (a) culturing cells that express a definitive endodermal        phenotype with about 1 ng/ml to about 100 ng/ml bFGF and about 5        ng/ml to about 500 ng/ml BMP4.

17. The method of statement 16, wherein the cells are cultured withabout 10 ng/ml bFGF and about 50 ng/ml BMP4.

18. A method for inducing cells that express a definitive endodermalphenotype into cells that express a liver-committed endodermal phenotypeand then into cells that express a hepatoblast phenotype, comprising:

-   -   (a) culturing cells that express a definitive endodermal        phenotype with about 1 ng/ml to about 100 ng/ml bFGF and about 5        ng/ml to about 500 ng/ml BMP4; and    -   (b) then culturing the cells of step (a) with about 5 ng/ml to        about 500 ng/ml aFGF, about 1 ng/ml to about 100 ng/ml FGF4 and        about 2.5 ng/ml to about 250 ng/ml FGF8b.

19. The method of statement 18, wherein the cells in step (a) arecultured with about 10 ng/ml bFGF and about 50 ng/ml BMP4, and the cellsin step (b) are cultured with about 50 ng/ml aFGF, about 10 ng/ml FGF4and about 25 ng/ml FGF8b.

20. A method for inducing cells that express a definitive endodermalphenotype into cells that express a liver-committed phenotype and theninto cells that express a hepatoblast phenotype and then into cells thatexpress a hepatocyte phenotype, comprising:

-   -   (a) culturing cells that express a definitive endodermal        phenotype with about 1 ng/ml to about 100 ng/ml bFGF and about 5        ng/ml to about 500 ng/ml BMP4;    -   (b) then culturing the cells of step (a) with about 5 ng/ml to        about 500 ng/ml aFGF, about 1 ng/ml to about 100 ng/ml FGF4 and        about 2.5 ng/ml to about 250 ng/ml FGF8b; and    -   (c) then culturing the cells of step (c) with about 2 ng/ml to        about 200 ng/ml HGF and about 10 ng/ml to about 1,000 ng/ml        Follistatin.

21. The method of statement 20, wherein the cells in step (a) arecultured with about 10 ng/ml bFGF and about 50 ng/ml BMP4, the cells instep (b) are cultured with about 50 ng/ml aFGF, about 10 ng/ml FGF4 andabout 25 ng/ml FGF8b, and the cells in step (c) are cultured with about20 ng/ml HGF and about 100 ng/ml Follistatin.

22. A method for inducing cells that express a liver-committedendodermal phenotype to differentiate into cells that express ahepatoblast phenotype, comprising:

-   -   (a) culturing cells that express a liver-committed endodermal        phenotype with about 5 ng/ml to about 500 ng/ml aFGF, about 1        ng/ml to about 100 ng/ml FGF4 and about 2.5 ng/ml to about 250        ng/ml FGF8b.

23. The method of statement 22, wherein the cells are cultured withabout 50 ng/ml aFGF, about 10 ng/ml FGF4 and about 25 ng/ml FGF8b.

24. A method for inducing cells that express a liver-committed phenotypeinto cells that express a hepatoblast phenotype and then into cells thatexpress a hepatocyte phenotype, comprising:

-   -   (a) culturing cells that express a liver-committed phenotype        with about 5 ng/ml to about 500 ng/ml aFGF, about 1 ng/ml to        about 100 ng/ml FGF4 and about 2.5 ng/ml to about 250 ng/ml        FGF8b; and    -   (b) then culturing the cells of step (a) with about 2 ng/ml to        about 200 ng/ml HGF and about 10 ng/ml to about 1,000 ng/ml        Follistatin.

25. The method of statement 24, wherein the cells in step (a) arecultured with about 50 ng/ml aFGF, about 10 ng/ml FGF4 and about 25ng/ml FGF8b, and the cells in step (b) are cultured with about 20 ng/mlHGF and about 100 ng/ml Follistatin.

26. A method for inducing cells that express a hepatoblast phenotypeinto cells that express a hepatocyte phenotype, comprising:

-   -   (a) culturing cells that express a hepatoblast phenotype with        about 2 ng/ml to about 200 ng/ml HGF and about 10 ng/ml to about        1,000 ng/ml Follistatin.

27. The method of statement 26, wherein the cells are cultured withabout 20 ng/ml HGF and about 100 ng/ml Follistatin.

28. The methods herein, wherein the cells are cultured at one or moresteps in a medium containing a serum concentration ranging from 0% toabout 2%.

29. The method of statement 28, wherein the cells are cultured at one ormore steps in a medium containing a serum concentration of about 2%.

30. The methods herein, wherein the cells are cultured at one or moresteps in a medium containing about 10⁻⁴M to about 10⁻⁷ M dexamethasone.

31. The method of statement 30, wherein the cells are cultured at one ormore steps in a medium containing about 10⁻⁶M dexamethasone.

32. The methods herein, wherein the cells are cultured at one or moresteps for at least four days.

33. The method of statements herein, wherein the cells that express aprimitive endodermal phenotype are cultured for about six days, thecells that express a definitive endodermal phenotype are cultured forabout four days, the cells that express a liver-committed endodermalphenotype are cultured for about four days, and the cells that express ahepatoblast phenotype are cultured for about seven days.

34. The methods herein, wherein the cells are mammalian.

35. The method of statement 34, wherein the cells are human, mouse, orrat.

36. The methods herein, wherein the cells that are contacted with Wnt3Aand Activin A are embryonic stem cells or cells that are not embryonicstem cells, embryonic germ cells or germ cells, and can differentiateinto at least one cell type of each of the endodermal, ectodermal andmesodermal embryonic lineages.

37. The method of statement 36, wherein the cells are not embryonic germcells, embryonic stem cells or germ cells, and can differentiate into atleast one cell type of each of the endodermal, ectodermal and mesodermalembryonic lineages.

38. The method of statement 36, wherein the cells are embryonic stemcells.

39. The method of statement 37, wherein the cells are IPS cells.

40. The method of statement 37, wherein the cells are isolated from bonemarrow, placenta, umbilical cord, muscle, brain, liver spinal cord,blood or skin. But they can be derived from any tissue or cell, and alsoby de-differentiation.

41. Cells produced according to any one of the methods recited in thepreceding statements.

42. A composition, comprising cells that express a primitive endodermalphenotype in a culture medium comprising about 5 ng/ml to about 500ng/ml Wnt3a and about 10 ng/ml to about 1,000 ng/ml ActivinA.

43. The composition of statement 42, wherein the medium is comprised ofabout 50 ng/ml Wnt3a and about 100 ng/ml ActivinA.

44. A composition, comprising cells that express a definitive endodermalphenotype in a culture medium comprising about 1 ng/ml to about 100ng/ml bFGF and about 5 ng/ml to about 500 ng/ml BMP4.

45. The composition of statement 44, wherein the medium is comprised ofabout 10 ng/ml bFGF and about 50 ng/ml BMP4.

46. A composition, comprising cells that express a liver-committedendodermal phenotype in a culture medium comprising about 5 ng/ml toabout 500 ng/ml aFGF, about 1 ng/ml to about 100 ng/ml FGF4 and about2.5 ng/ml to about 250 ng/ml FGF8b.

47. The composition of statement 46, wherein the medium is comprised ofabout 50 ng/ml aFGF, about 10 ng/ml FGF4 and about 25 ng/ml FGF8b.

48. A composition, comprising cells that express a hepatoblast phenotypein a culture medium comprising about 2 ng/ml to about 200 ng/ml HGF andabout 10 ng/ml to about 1,000 ng/ml Follistatin.

49. The composition of statement 48, wherein the medium is comprised ofabout 20 ng/ml HOF and about 100 ng/ml Follistatin.

50. The compositions herein, wherein the medium further comprises serumin a concentration from 0% to about 2%.

51. The composition of statement 50, wherein the serum concentration isabout 2%.

52. The compositions herein, wherein the medium further comprises about10⁻⁴ M to about 10⁻⁷ M dexamethasone.

53. The composition of statement 52, wherein the concentration ofdexamethasone is about 10⁻⁶ M.

54. The compositions herein, wherein the cells are mammalian.

55. The composition of statement 54, wherein the cells are human.

56. The compositions herein, wherein the cells are embryonic stem cellsor cells that are not embryonic stem cells, embryonic germ cells or germcells, and can differentiate into at least one cell type of each of theendodermal, ectodermal and mesodermal embryonic lineages.

57. The composition of statement 56, wherein the cells are not embryonicgerm cells, embryonic stem cells or germ cells, and can differentiateinto at least one cell type of each of the endodermal, ectodermal andmesodermal embryonic lineages.

58. The composition of statement 56, wherein the cells are embryonicstem cells.

59. The composition of statement 57, wherein the cells are IPS cells.

60. The composition of statement 57, wherein the cells are isolated frombone marrow, placenta, umbilical cord, muscle, brain, liver spinal cord,blood or skin.

In one embodiment, the serum is fetal bovine serum.

In one embodiment, culturing cells with Wnt3a is about 2.5 days with arange of about 1.5-3.5 days, such as 2 or 3 days.

Any cell can be used in the initial step of culture with Wnt3a andActivin A as long as it has a phenotype of a cell that is prior to theprimitive streak. Such a cell could express Oct3/4. For an embryonicstem cell, for example, the phenotype would be inner cell mass cell orepiblast. Cells include, but are not limited to, primordial germ cells,embryonic germ cells, cells produced by somatic cell nucleartransplantation into oocytes, tumor cell lines, embryonal carcinomacells, blastomere cells, inner cell mass cells, embryonic stem cellcultures and lines, spermatogonial stem cells, epliblast cells, andother non-embryonic stem cells, such as reprogrammed somatic cells(IPSC). In one embodiment, such cells express Oct3/4 at levels greaterthan about 0.1% of Oct3/4 expression in embryonic stem cells.

Cells at any step (after the initial step with Wnt3a and Activin A) canbe produced by the methods of the invention or can be derived from an invivo or in vitro source. For example, definitive endodermal cells can bederived from in vivo and cultured according to the conditions describedherein to produce cells with a liver-committed phenotype, cells with aliver-committed phenotype can be derived from in vivo and culturedaccording to the conditions described herein to produce cells with ahepatoblast phenotype, etc.

61. A pharmaceutical composition comprising the cells produced accordingto any one of the methods herein.

62. A method of treatment comprising administering a therapeuticallyeffective amount of the cells produced according to any one of themethods herein to a subject with a liver deficiency.

The invention is also directed to methods of using the cells produced bythe methods for treatment of liver deficiencies.

The invention is also directed to methods of using the cells for studiesof liver metabolism, for example, to identify or assess metabolicmodulators.

The invention is also directed to methods of using the cells for studiesof liver toxicity, for example, to identify or assess the toxicity ofspecific compounds.

The invention is also directed to pharmaceutical compositions containingthe cells of the invention. Such compositions are suitable foradministration to subjects in need of such cells. The cells would beadministered in therapeutically effective amounts.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides the 4-step in vitro differentiation protocol. Days 0-6:Activin A (100 ng/ml) and Wnt3a (50 ng/ml). Days 6-10: bFGF (10 ng/ml)and BMP4 (50 ng/ml). Days 10-14: aFGF (50 ng/ml), FGF4 (10 ng/ml) andFGF8b (25 ng/ml). Days 14-21: HGF (20 ng/ml) and Follistatin (100ng/ml).

FIG. 2 are light micrographs showing an overview of the changes inmorphology of differentiating HO-rMAPCs at different timepoints ofdifferentiation.

FIG. 3 are light micrographs showing the morphological changes observedduring differentiation of rMAPC in differentiation medium withoutcytokine supplementation and changes in morphology of differentiatingHO-rMAPCs.

FIG. 4 A-F are electron micrographs showing characteristics of fullydifferentiated HO-rMAPCs.

FIG. 5 A-F shows immunohistochemical staining for rAFP at differenttimepoints of differentiation of rMAPC: A=day 3; B=day 6; C=day 12;D=day 16; E=day 20; and F=isotype control.

FIG. 6 A-F shows immunohistochemical staining for rALB at differenttimepoints of differentiation of rMAPCs.

FIG. 7 shows immunohistochemical staining for CK18 at differenttimepoints of differentiation of rMAPCs. The top three lines are,respectively, undifferentiated rMAPC-1; after day 20; and in mature rathepatocytes. The lower three lines show the respective isotype controls.

FIG. 8 is a graph illustrating the time-dependent increase of albuminconcentration during hepatic differentiation of rMAPCs (mean of >10experiments).

FIG. 9 is a graph illustrating glycogen storage (μg/mg protein) in rMAPC(n=6-10).

FIG. 10 A-B are graphs illustrating the increasing formation ofmonoconjugated bilirubin after 4, 8, 12, 22, and 33 hours in mature rathepatocytes (A) and in differentiated rMAPC after day 20 (B). (n=3).

FIG. 11 A-J shows the expression of genes, using RT-qPCR, as well asvarious markers of differentiation, at different timepoints ofdifferentiation, of the hESC line H9. A=PS phenotype; B=ME/DE phenotype;C=hepatoblast phenotype; D=definitive hepatocyte phenotype;E=concentration of albumin; F=spontaneous production of urea andconversion of HN₄HCO₃ to urea; G=glycogen storage at differenttimepoints of differentiation; H=metabolization of differentconcentrations of unconjugated bilirubin at different timepoints ofdifferentiation; I=total GST activity at different timepoints ofdifferentiation; and J=expression of coagulation factors Factor V,Factor VII, Protein C, GGCX and VKOR at different timepoints ofdifferentiation.

FIG. 12 shows the albumin secreted by hiPS cells during differentiation.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

“A” or “an” means one or more than one.

“Comprising” means, without other limitation, including the referent,necessarily, without any qualification or exclusion on what else may beincluded. For example, “a composition comprising x and y” encompassesany composition that contains x and y, no matter what other componentsmay be present in the composition. Likewise, “a method comprising thestep of x” encompasses any method in which x is carried out, whether xis the only step in the method or it is only one of the steps, no matterhow many other steps there may be and no matter how simple or complex xis in comparison to them. “Comprised of” and similar phrases using wordsof the root “comprise” are used herein as synonyms of “comprising” andhave the same meaning.

“Definitive endodermal phenotype” is a particular phenotype of cellsthat no longer express the self-renewal gene Oct3/4, do not express theprimitive endoderm gene Sox7, do not express the mesodermal gene Flk1,but express Sox17, Foxa2, E-cadherin, CXCR4, and PDGF-Ra.

“Effective amount” generally means an amount which provides the desiredlocal or systemic effect. For example, an effective amount is an amountsufficient to effectuate a beneficial or desired clinical result. Theeffective amounts can be provided all at once in a single administrationor in fractional amounts that provide the effective amount in severaladministrations. The precise determination of what would be consideredan effective amount may be based on factors individual to each subject,including their size, age, injury, and/or disease or injury beingtreated, and amount of time since the injury occurred or the diseasebegan. One skilled in the art will be able to determine the effectiveamount for a given subject based on these considerations which areroutine in the art. As used herein, “effective dose” means the same as“effective amount.”

“EC cells” were discovered from analysis of a type of cancer called ateratocarcinoma. In 1964, researchers noted that a single cell interatocarcinomas could be isolated and remain undifferentiated inculture. This type of stem cell became known as an embryonic carcinomacell (EC cell).

“Embryonic Stem Cells (ESC)” are well known in the art and have beenprepared from many different mammalian species for many years. Embryonicstem cells are stem cells derived from the inner cell mass of an earlystage embryo known as a blastocyst. They are able to differentiate intoall derivatives of the three primary germ layers: ectoderm, endoderm,and mesoderm. These include each of the more than 220 cell types in theadult body. The ES cells can become any tissue in the body, excludingplacenta. Only the morula's cells are totipotent, able to become alltissues and a placenta.

“Hepatic differentiation factors” are chemical or biological factorsthat induce differentiation of stem and progenitor cells into moredifferentiated cells of the hepatic lineage. Hepatic differentiationfactors include, but are not limited to, Wnt3a, ActivinA, bFGF, BMP4,aFGF, FGF4, FGF8b, HGF and Follistatin. The initial cell may expressOct3/4.

“Hepatoblast phenotype” is a particular phenotype of cells thatco-express albumin, alpha fetoprotein and keratin 19, and express, onthe cell membrane, c-Met, EPCAM, and Dlk1 (Tanimizu, N. et al., J CellSci, 116:1775-1786 (2003)).

“Hepatocyte phenotype” is a particular phenotype of cells that expressalbumin and keratin 18 but not alpha fetoprotein and keratin 19; inaddition, hepatocytes may express one or more of TAT, MRP2, G6P, GLYS2,PEPCK, A1AT, BSEP, CX-32, NTCP, CYP7A1 (rat) and CYP3A4 (human).

Use of the term “includes” is not intended to be limiting. For example,stating that an inhibitor “includes fragments and variants does not meanthat other forms of the inhibitor are excluded.

“Induced pluripotent stem cells (IPSC or IPS cells)” are somatic cellsthat have been reprogrammed for example, by introducing exogenous genesthat confer on the somatic cell a less differentiated phenotype. Thesecells can then be induced to differentiate into less differentiatedprogeny. IPS cells have been derived using modifications of an approachoriginally discovered in 2006 (Yamanaka, S. et al., Cell Stem Cell,1:39-49 (2007)). For example, in one instance, to create IPS cells,scientists started with skin cells that were then modified by a standardlaboratory technique using retroviruses to insert genes into thecellular DNA. In one instance, the inserted genes were Oct4, Sox2, Lif4,and c-myc, known to act together as natural regulators to keep cells inan embryonic stem cell-like state. These cells have been described inthe literature. See, for example, Wernig et al., PNAS,105:5856-5861(2008); Jaenisch et al., Cell, 132:567-582 (2008); Hanna etal., Cell, 133:250-264 (2008); and Brambrink et al., Cell Stem Cell,2:151-159 (2008). These references are incorporated by reference forteaching IPSCs and methods for producing them. It is also possible thatsuch cells can be created by specific culture conditions (exposure tospecific agents).

The term “isolated” refers to a cell or cells that are not associatedwith one or more cells or one or more cellular components that areassociated with the cell or cells in vivo. An “enriched population”means a relative increase in numbers of a desired cell relative to oneor more other cell types in vivo or in primary culture.

However, as used herein, the term “isolated” does not indicate thepresence of only a specific desired cell, such as a stem or hepaticprogenitor cell. Rather, the term “isolated” indicates that the cellsare removed from their natural tissue environment and are present at ahigher concentration as compared to the normal tissue environment.Accordingly, an “isolated” cell population may further include celltypes in addition to stem cells and may include additional tissuecomponents. This also can be expressed in terms of cell doublings, forexamples. A cell may have undergone 10, 20, 30, 40 or more doublings invitro or ex vivo so that it is enriched compared to its original numbersin vivo or in its original tissue environment (e.g., bone marrow,peripheral blood, adipose tissue, etc.)

“Liver-committed endodermal phenotype” is a particular phenotype ofcells that are EPCAM positive and Dlk1 Negative (Tanimizu, N. et al., JCell Sci, 116:1775-1786 (2003)).

“MAPC” is an acronym for “multipotent adult progenitor cell.” It refersto a non-embryonic stem cell that can give rise to cell lineages of allthree germ layers (i.e., endoderm, mesoderm and ectoderm) upondifferentiation. Like embryonic stem cells, human MAPCs expresstelomerase, Oct 3/4 (i.e., Oct 3A), rex-1, rox-1 and sox-2, and mayexpress SSEA-4. The term “adult” in MAPC is non-restrictive. It refersto a non-embryonic somatic cell.

MAPCs constitutively express Oct 3/4 and high levels of telomerase(Jiang, Y. et al., Nature, 418:41(2002); Exp Hematol, 30:896, 2002).MAPCs derived from human, mouse, rat or other mammals appear to be theonly normal, non-malignant, somatic cell (i.e., non-germ cell) known todate to express very high levels of telomerase even in late passagecells. The telomeres are extended in MAPCs and they are karyotypicallynormal. Because MAPCs injected into a mammal can migrate to andassimilate within multiple organs, MAPCs are self-renewing stem cells.

“Multipotent,” with respect to the term in “MAPC,” refers to the abilityto give rise to cell lineages of more than one primitive germ layer(i.e., endoderm, mesoderm and ectoderm) upon differentiation, such asall three. This term is not used consistently in the literature.

“Pluripotent” as used herein means any cell that, when exposed to Wnt3aand Activin A at the specified amounts, gives rise to cells with adefinitive endodermal phenotype. Such cells may have the ability to giverise to cell lineages of more than one primitive germ layer (i.e.,endoderm, mesoderm and ectoderm) upon differentiation, such as allthree.

“Primitive endodermal phenotype” is a particular phenotype of cells thatmay express sox7, sox17, gata4, gata6, Cited1, Tcf2, Lamb1, Dab2, LamAl,LamA4, Lamc1, Col4a1, and Nidogen2 (this is a phenotype of mouse and ratMAPC, XEN cells from J. Rossant and Sox7 expressing ESC from J. Rossant.See also Ulloa-Montoya et al., Genome Biol, 8:R163 (2007); Se'guin etal., Cell Stem Cell, 3:182-195 (2008); and Kunath et al., Development,132:1649-1661(2005)).

“Primordial embryonic germ cells” (PG or EG cells) can be cultured andstimulated to produce many less differentiated cell types.

“Progenitor cells” are cells produced during differentiation of a stemcell that have some, but not all, of the characteristics of theirterminally-differentiated progeny. The term “progenitor” as used in theacronym “MAPC” does not limit these cells to a particular lineage. Ahepatocyte progenitor is any cell in the hepatocyte lineage that is lessdifferentiated than a hepatocyte.

“Self-renewal” refers to the ability to produce replicate daughter stemcells having differentiation potential that is identical to those fromwhich they arose. A similar term used in this context is“proliferation.”

“Stem cell” means a cell that can undergo self-renewal (i.e., progenywith the same differentiation potential) and also produce progeny cellsthat are more restricted in differentiation potential. Within thecontext of the invention, a stem cell would also encompass a moredifferentiated cell that has dedifferentiated, for example, by nucleartransfer, by fusions with a more primitive stem cell, by introduction ofspecific transcription factors, or by culture under specific conditions.See, for example, Wilmut et al., Nature, 385:810-813 (1997); Ying etal., Nature, 416:545-548 (2002); Guan et al., Nature, 440:1199-1203(2006); Takahashi et al., Cell, 126:663-676 (2006); Okita et al.,Nature, 448:313-317 (2007); and Takahashi et al., Cell, 131:861-872(2007).

Dedifferentiation may also be caused by the administration of certaincompounds or exposure to a physical environment in vitro or in vivo thatwould cause the dedifferentiation. Stem cells also may be derived fromabnormal tissue, such as a teratocarcinoma and some other sources suchas embryoid bodies (although these can be considered embryonic stemcells in that they are derived from embryonic tissue, although notdirectly from the inner cell mass).

“Subject” means a vertebrate, such as a mammal, such as a human. Mammalsinclude, but are not limited to, humans, dogs, cats, horses, cows andpigs.

The term “therapeutically effective amount” refers to the amountdetermined to produce any therapeutic response in a mammal. For example,effective amounts of the therapeutic cells or cell-associated agents mayprolong the survivability of the patient, and/or inhibit overt clinicalsymptoms. Treatments that are therapeutically effective within themeaning of the term as used herein, include treatments that improve asubject's quality of life even if they do not improve the diseaseoutcome per se. Such therapeutically effective amounts are ascertainedby one of ordinary skill in the art through routine application tosubject populations such as in clinical and pre-clinical trials. Thus,to “treat” means to deliver such an amount.

“Treat,” “treating” or “treatment” are used broadly in relation to theinvention and each such term encompasses, among others, preventing,ameliorating, inhibiting, or curing a deficiency, dysfunction, disease,or other deleterious process, including those that interfere with and/orresult from a therapy.

Methods and Compositions of the Invention

The methods of the invention induce cells in culture to progress throughthe appropriate stages of hepatic development, thus recapitulatinghepatic development in vitro and, as a result, give rise to cells havingfunctional hepatic properties (e.g., biochemical and anatomicalcharacteristics of hepatic cells).

Culture methods of the invention comprise a sequential addition ofhepatic differentiation factors to cells, wherein there is a firstaddition of about 5 ng/ml to about 500 ng/ml Wnt3a, more particularlyabout 50 ng/ml Wnt3a, and about 10 ng/ml to about 1,000 ng/ml ActivinA,more particularly about 100 ng/ml ActivinA; a second addition of about 1ng/ml to about 100 ng/ml bFGF, more particularly about 10 ng/ml bFGF,and 5 ng/ml to about 500 ng/ml BMP4, more particularly about 50 ng/mlBMP4; a third addition of 5 ng/ml to about 500 ng/ml aFGF, moreparticularly about 50 ng/ml aFGF, about 1 ng/ml to about 100 ng/ml FGF4,more particularly about 10 ng/ml FGF4, and about 2.5 ng/ml to about 250ng/ml FGF8b, more particularly about 25 ng/ml FGF8b; and a fourthaddition of about 2 ng/ml to about 200 ng/ml HGF, more particularlyabout 20 ng/ml HGF, and about 10 ng/ml to about 1,000 ng/ml Follistatin,more particularly about 100 ng/ml Follistatin.

At each successive step, the culture is continued for at least fourdays. More particularly, the cells are cultured in the first step forabout six days; in the second step for about four days; in the thirdstep for about four days; and in the fourth step for about seven days.In one embodiment, cells are cultured with Wnt3a for about 2.5 days.

At one or more steps, the cells are cultured in a medium containing aserum concentration from 0% to about 2%, more particularly about 2%.

Additionally, at one or more steps, the cells are cultured in a mediumcontaining about 10⁻⁴ M to about 10⁻⁷ M dexamethasone, more particularlyabout 10⁻⁶ M dexamethasone.

Culture medium at each successive step of the methods of the presentinvention is prepared to contain only the growth factor(s) describedabove, and cells are washed between each step to reduce the presence ofpreviously added growth factor(s). Alternatively, reduced concentrationsof the previously provided factor(s) in a previous step can remain inthe culture medium of the next step.

The methods of the present invention contemplate the use of any Wnt3a,ActivinA, bFGF, BMP4, aFGF, FGF4, FGF8b, HGF and Follistatin known inthe art and having conserved function, and from all species (e.g.,orthologs from human, mouse, rat, monkey, pig and the like). The hepaticdifferentiation factors of the present invention are well known to thoseskilled in the art.

Suitable forms of Wnt3a, ActivinA, bFGF, BMP4, aFGF, FGF4, FGF8b, HGFand Follistatin include, but are not limited to, isolated polypeptides,which are optionally recombinant, including whole proteins, partialproteins (e.g., domains) and peptide fragments. Fragments of apolypeptide preferably are those fragments that retain the distinctfunctional capability of the particular factor, which in the presentinvention generally relates to the ability to influence hepaticdifferentiation (the specific function of each factor is well known inthe art). Such polypeptides also include, but are not limited to, fusionproteins and chimeric proteins. Short polypeptides can be synthesizedchemically using well-established methods of peptide synthesis.

Cytokines may be replaced by small molecules that activate the samesignal pathway, such as GSK3b inhibitor for Wnt3a; kinase activatingmolecules for the FGFs.

The culture methods of the present invention comprise a sequentialaddition of hepatic differentiation factors to cells.

In the first step of the present invention, the hepatic differentiationfactors Wnt3a and Activin A are added to the cells.

The concentration of Wnt3a that is added to the cells can range fromabout 5 ng/ml to about 500 ng/ml. However, the invention alsoencompasses sub-ranges of concentrations of Wnt3a. For example, fromabout 5-25 ng/ml, 25-50 ng/ml, 50-75 ng/ml, 75-100 ng/ml, 100-150 ng/ml,150-300 ng/ml and 300-500 ng/ml. The preferred concentration of Wnt3athat is added to the cells is about 50 ng/ml. The duration of Wnt3aexposure used in the examples is six days. However, this may be changedto two, three, four, or five days.

The concentration of Activin A that is added to the cells can range fromabout 10 ng/ml to about 1000 ng/ml. However, the invention alsoencompasses sub-ranges of concentrations of Activin A. For example, fromabout 10-25 ng/ml, 25-50 ng/ml, 50-75 ng/ml, 75-100 ng/ml, 100-125ng/ml, 125-150 ng/ml, 150-175 ng/ml, 175-200 ng/ml, 200-400 ng/ml,400-600 ng/ml, 600-800 ng/ml and 800-1000 ng/ml. The preferredconcentration of Activin A that is added to the cells is about 100ng/ml. The duration of Activin A exposure used in the examples is sixdays. However, this may be changed to four, five, or seven days.

In the second step of the present invention, the hepatic differentiationfactors bFGF and BMP4 are added to the cells.

The concentration of bFGF that is added to the cells can range fromabout 1 ng/ml to about 100 ng/ml. However, the invention alsoencompasses sub-ranges of concentrations of bFGF. For example, fromabout 1-2 ng/ml, 2-4 ng/ml, 4-6 ng/ml, 6-8 ng/ml, 8-10 ng/ml, 10-12ng/ml, 12-14 ng/ml, 14-16 ng/ml, 16-18 ng/ml, 18-20 ng/ml, 20-40 ng/ml,40-60 ng/ml, 60-80 ng/ml and 80-100 ng/ml. The preferred concentrationof bFGF that is added to the cells is about 10 ng/ml. The duration ofbFGF exposure used in the examples is five days. However, this may bechanged to four, six, or seven days.

The concentration of BMP4 that is added to the cells can range fromabout 5 ng/ml to about 500 ng/ml. However, the invention alsoencompasses sub-ranges of concentrations of BMP4. For example, fromabout 5-10 ng/ml, 10-20 ng/ml, 20-30 ng/ml, 30-40 ng/ml, 40-50 ng/ml,50-60 ng/ml, 60-70 ng/ml, 70-80 ng/ml, 80-90 ng/ml, 90-100 ng/ml,100-200 ng/ml, 200-300 ng/ml, 300-400 ng/ml and 400-500 ng/ml. Thepreferred concentration of BMP4 that is added to the cells is about 50ng/ml. The duration of BMP4 exposure used in the examples is five days.However, this may be changed to four, six, or seven days.

In the third step of the present invention, the hepatic differentiationfactors aFGF, FGF4 and FGF8b are added to the cells.

The concentration of aFGF that is added to the cells can range fromabout 5 ng/ml to about 500 ng/ml. However, the invention alsoencompasses sub-ranges of concentrations of aFGF. For example, fromabout 5-10 ng/ml, 10-20 ng/ml, 20-30 ng/ml, 30-40 ng/ml, 40-50 ng/ml,50-60 ng/ml, 60-70 ng/ml, 70-80 ng/ml, 80-90 ng/ml, 90-100 ng/ml,100-200 ng/ml, 200-300 ng/ml, 300-400 ng/ml and 400-500 ng/ml. Thepreferred concentration of aFGF that is added to the cells is about 50ng/ml. The duration of aFGF exposure used in the examples is five days.However, this may be changed to four, six, or seven days.

The concentration of FGF4 that is added to the cells can range fromabout 1 ng/ml to about 100 ng/ml. However, the invention alsoencompasses sub-ranges of concentrations of FGF4. For example, fromabout 1-2 ng/ml, 2-4 ng/ml, 4-6 ng/ml, 6-8 ng/ml, 8-10 ng/ml, 10-20ng/ml, 20-30 ng/ml, 30-40 ng/ml, 40-60 ng/ml, 60-80 ng/ml and 80-100ng/ml. The preferred concentration of FGF4 that is added to the cells isabout 10 ng/ml. The duration of FGF4 exposure used in the examples isfive days. However, this may be changed to four, six, or seven days.

The concentration of FGF8b that is added to the cells can range fromabout 2.5 ng/ml to about 250 ng/ml. However, the invention alsoencompasses sub-ranges of concentrations of FGF8b. For example, fromabout 2.5-5 ng/ml, 5-10 ng/ml, 10-15 ng/ml, 15-20 ng/ml, 20-25 ng/ml,25-30 ng/ml, 35-40 mg/ml, 45-50 ng/ml, 50-100 ng/ml, 100-150 ng/ml,150-200 ng/ml and 200-250 ng/ml. The preferred concentration of FGF8bthat is added to the cells is about 25 ng/ml. The duration of FGF8bexposure used in the examples is five days. However, this may be changedto four, six, or seven days.

In the fourth step of the present invention, the hepatic differentiationfactors HGF and Follistatin are added to the cells.

The concentration of HGF that is added to the cells can range from about2 ng/ml to about 200 ng/ml. However, the invention also encompassessub-ranges of concentrations of HGF. For example, from about 2-5 ng/ml,5-10 ng/ml, 10-15 ng/ml, 15-20 ng/ml, 20-25 ng/ml, 25-30 ng/ml, 30-35ng/ml, 35-40 ng/ml, 40-50 ng/ml, 50-100 ng/ml, 100-150 ng/ml and 150-200ng/ml. The preferred concentration of HGF that is added to the cells isabout 20 ng/ml. The duration of HGF exposure used in the examples isfive days. However, this may be changed to four, six, or seven days andcan be as high as 30 days.

The concentration of Follistatin that is added to the cells can rangefrom about 10 ng/ml to about 1000 ng/ml. However, the invention alsoencompasses sub-ranges of concentrations of Follistatin. For example,from about 10-25 ng/ml, 25-50 ng/ml, 50-75 ng/ml, 75-100 ng/ml, 100-125ng/ml, 125-150 ng/ml, 150-175 ng/ml, 150-175 ng/ml, 175-200 ng/ml,200-400 ng/ml, 400-600 ng/ml, 600-800 ng/ml and 800-1000 ng/ml. Thepreferred concentration of Follistatin that is added to the cells isabout 100 ng/ml. The duration of Follistatin exposure used in theexamples is five days. However, this may be changed to four, six, orseven days and can be as high as 30 days.

Stem Cells

The present invention can be practiced, preferably, using stem cells ofvertebrate species, such as humans, non-human primates, domesticanimals, livestock, and other non-human mammals.

Embryonic

Stem cells have been identified in most tissues. The most well studiedstem cell is the embryonic stem cell (ESC), as it has unlimitedself-renewal and multipotent differentiation potential. These cells arederived from the inner cell mass of the blastocyst or can be derivedfrom the primordial germ cells of a post-implantation embryo (embryonalgerm cells or EG cells). ES and EG cells have been derived, first frommouse, and later, from many different animals, and more recently, fromnon-human primates and humans. When introduced into mouse blastocysts orblastocysts of other animals, ESCs can contribute to all tissues of theanimal. ES and EG cells can be identified by positive staining withantibodies against SSEA1 (mouse) and SSEA4 (human). See, for example,U.S. Pat. Nos. 5,453,357; 5,656,479; 5,670,372; 5,843,780; 5,874,301;5,914,268; 6,110,739 6,190,910; 6,200,806; 6,432,711; 6,436,701,6,500,668; 6,703,279; 6,875,607; 7,029,913; 7,112,437; 7,145,057;7,153,684; and 7,294,508, each of which is incorporated by referenceherein for teaching ESCs and methods of making and expanding ESCs.Accordingly, ESCs and methods for isolating and expanding ESCs arewell-known in the art.

A number of transcription factors and exogenous cytokines have beenidentified that influence the potency status of embryonic stem cells invivo. The first transcription factor to be described that is involved instem cell pluripotency is Oct4. Oct4 belongs to the POU (Pit-Oct-Unc)family of transcription factors and is a DNA binding protein that isable to activate the transcription of genes, containing an octamericsequence called “the octamer motif” within the promotor or enhancerregion. Oct4 is expressed at the moment of the cleavage stage of thefertilized zygote until the egg cylinder is formed. The function ofOct3/4 is to repress differentiation inducing genes (i.e., FoxaD3, hCG)and to activate genes promoting pluripotency (FGF4, Utf1, Rex1). Sox2, amember of the high mobility group (HMG) box transcription factors,cooperates with Oct4 to activate transcription of genes expressed in theinner cell mass. It is essential that Oct3/4 expression in embryonicstem cells is maintained between certain levels. Overexpression ordownregulation of >50% of Oct4 expression level will alter embryonicstem cell fate, with the formation of primitive endoderm/mesoderm ortrophectoderm, respectively. In vivo, Oct4 deficient embryos develop tothe blastocyst stage, but the inner cell mass cells are not pluripotent.Instead they differentiate along the extraembryonic trophoblast lineage.Sall4, a mammalian Spalt transcription factor, is an upstream regulatorof Oct4, and is therefore important to maintain appropriate levels ofOct4 during early phases of embryology. When Sall4 levels fall below acertain threshold, trophectodermal cells will expand ectopically intothe inner cell mass. Another transcription factor required forpluripotency is Nanog, named after a celtic tribe “Tir Nan Og”: the landof the ever young. In vivo, Nanog is expressed from the stage of thecompacted morula, is subsequently defined to the inner cell mass and isdownregulated by the implantation stage. Downregulation of Nanog may beimportant to avoid an uncontrolled expansion of pluripotent cells and toallow multilineage differentiation during gastrulation. Nanog nullembryos, isolated at day 5.5, consist of a disorganized blastocyst,mainly containing extraembryonic endoderm and no discernable epiblast.

Non-Embryonic

An example of a non-embryonic stem cell is adipose-derived adult stemcells (ADSCs) which have been isolated from fat, typically byliposuction followed by release of the ADSCs using collagenase. ADSCsare similar in many ways to MSCs derived from bone marrow, except thatit is possible to isolate many more cells from fat. These cells havebeen reported to differentiate into bone, fat, muscle, cartilage andneurons. A method of isolation has been described in U.S. 2005/0153442.

Other non-embryonic cells reported to be capable of differentiating intocell types of more than one embryonic germ layer include, but are notlimited to, cells from umbilical cord blood (see U.S. Publication No.2002/0164794), placenta (see U.S. Publication No. 2003/0181269;umbilical cord matrix (Mitchell, K. E. et al., Stem Cells, 21:50-60,2003), small embryonic-like stem cells (Kucia, M. et al., J PhysiolPharmacol, 57 Suppl 5:5-18, 2006), amniotic fluid stem cells (Atala, A.,J Tissue Regen Med, 1:83-96, 2007), skin-derived precursors (Toma etal., Nat Cell Biol, 3:778-784, 2001), and bone marrow (see U.S.Publication Nos. 2003/0059414 and 2006/0147246), each of which isincorporated by reference herein for teaching these cells.

Other stem cells that are known in the art include gastrointestinal stemcells, epidermal stem cells, and hepatic stem cells, which also havebeen termed “oval cells” (Potten, C., et al., Trans R Soc Lond B BiolSci, 353:821-830 (1998);, Watt, F., Trans R Soc Lond B Biol Sci,353:831(1997); Alison et al., Hepatology, 29:678-683 (1998).

Strategies of Reprogramming Somatic Cells

Several different strategies such as nuclear transplantation, cellularfusion, and culture induced reprogramming have been employed to inducethe conversion of differentiated cells into an embryonic state. Nucleartransfer involves the injection of a somatic nucleus into an enucleatedoocyte, which, upon transfer into a surrogate mother, can give rise to aclone (“reproductive cloning”), or, upon explantation in culture, cangive rise to genetically matched embryonic stem (ES) cells (“somaticcell nuclear transfer,” SCNT). Cell fusion of somatic cells with EScells results in the generation of hybrids that show all features ofpluripotent ES cells. Explantation of somatic cells in culture selectsfor immortal cell lines that may be pluripotent or multipotent. Atpresent, spermatogonial stem cells are the only source of pluripotentcells that can be derived from postnatal animals. Transduction ofsomatic cells with defined factors can initiate reprogramming to apluripotent state. These experimental approaches have been extensivelyreviewed (Hochedlinger and Jaenisch, Nature, 441:1061-1067 (2006) andYamanaka, S., Cell Stem Cell, 1:39-49 (2007)).

Nuclear Transfer

Nuclear transplantation (NT), also referred to as somatic cell nucleartransfer (SCNT), denotes the introduction of a nucleus from a donorsomatic cell into an enucleated ogocyte to generate a cloned animal suchas Dolly the sheep (Wilmut et al., Nature, 385:810-813 (1997). Thegeneration of live animals by NT demonstrated that the epigenetic stateof somatic cells, including that of terminally differentiated cells,while stable, is not irreversible fixed but can be reprogrammed to anembryonic state that is capable of directing development of a neworganism. In addition to providing an exciting experimental approach forelucidating the basic epigenetic mechanisms involved in embryonicdevelopment and disease, nuclear cloning technology is of potentialinterest for patient-specific transplantation medicine.

Fusion of Somatic Cells and Embryonic Stem Cells

Epigenetic reprogramming of somatic nuclei to an undifferentiated statehas been demonstrated in murine hybrids produced by fusion of embryoniccells with somatic cells. Hybrids between various somatic cells andembryonic carcinoma cells (Softer, D., Nat Rev Genet, 7:319-327 (2006),embryonic germ (EG), or ES cells (Zwaka and Thomson, Development,132:227-233 (2005)) share many features with the parental embryoniccells, indicating that the pluripotent phenotype is dominant in suchfusion products. As with mouse (Tada et al., Curr Biol, 11:1553-1558(2001)), human ES cells have the potential to reprogram somatic nucleiafter fusion (Cowan et al., Science, 309:1369-1373 (2005)); Yu et al.,Science, 318:1917-1920 (2006)). Activation of silent pluripotencymarkers such as Oct4 or reactivation of the inactive somatic Xchromosome provided molecular evidence for reprogramming of the somaticgenome in the hybrid cells. It has been suggested that DNA replicationis essential for the activation of pluripotency markers, which is firstobserved 2 days after fusion (Do and Scholer, Stem Cells, 22:941-949(2004)), and that forced overexpression of Nanog in ES cells promotespluripotency when fused with neural stem cells (Silva et al., Nature,441:997-1001(2006)).

Culture-Induced Reprogramming

Pluripotent cells have been derived from embryonic sources such asblastomeres and the inner cell mass (ICM) of the blastocyst (ES cells),the epiblast (EpiSC cells), primordial germ cells (EG cells), andpostnatal spermatogonial stem cells (“maGSCsm” “ES-like” cells). Thefollowing pluripotent cells, along with their donor cell/tissue is asfollows: parthogenetic ES cells are derived from murine oocytes(Narasimha et al., Curr Biol, 7:881-884 (1997)); embryonic stem cellshave been derived from blastomeres (Wakayama et al., Stem Cells,25:986-993 (2007)); inner cell mass cells (source not applicable) (Egganet al., Nature, 428:44-49 (2004)); embryonic germ and embryonalcarcinoma cells have been derived from primordial germ cells (Matsui etal., Cell, 70:841-847 (1992)); GMCS, maSSC, and MASC have been derivedfrom spermatogonial stem cells (Guan et al., Nature, 440:1199-1203(2006); Kanatsu-Shinohara et al., Cell, 119:1001-1012 (2004); andSeandel et al., Nature, 449:346-350 (2007)); EpiSC cells are derivedfrom epiblasts (Brons et al., Nature, 448:191-195 (2007); Tesar et al.,Nature, 448:196-199 (2007)); parthogenetic ES cells have been derivedfrom human oocytes (Cibelli et al., Science, 295L819 (2002); Revazova etal., Cloning Stem Cells, 9:432-449 (2007)); human ES cells have beenderived from human blastocysts (Thomson et al., Science, 282:1145-1147(1998)); MAPC have been derived from bone marrow (Jiang et al., Nature,418:41-49 (2002); Phinney and Prockop, Stem Cells, 25:2896-2902 (2007));cord blood cells (derived from cord blood) (van de Ven et al., ExpHematol, 35:1753-1765 (2007)); neurosphere derived cells derived fromneural cell (Clarke et al., Science, 288:1660-1663 (2000)). Donor cellsfrom the germ cell lineage such as PGCs or spermatogonial stem cells areknown to be unipotent in vivo, but it has been shown that pluripotentES-like cells (Kanatsu-Shinohara et al., Cell, 119:1001-1012 (2004) ormaGSCs (Guan et al., Nature, 440:1199-1203 (2006), can be isolated afterprolonged in vitro culture. While most of these pluripotent cell typeswere capable of in vitro differentiation and teratoma formation, onlyES, EG, EC, and the spermatogonial stem cell-derived maGCSs or ES-likecells were pluripotent by more stringent criteria, as they were able toform postnatal chimeras and contribute to the germline. Recently,multipotent adult spermatogonial stem cells (MASCs) were derived fromtesticular spermatogonial stem cells of adult mice, and these cells hadan expression profile different from that of ES cells (Seandel et al.,Nature, 449:346-350 (2007)) but similar to EpiSC cells, which werederived from the epiblast of postimplantation mouse embryos (Brons etal., Nature, 448:191-195 (2007); Tesar et al., Nature, 448:196-199(2007)).

Reprogramming by Defined Transcription Factors

Takahashi and Yamanaka have reported reprogramming somatic cells back toan ES-like state (Takahashi and Yamanaka, Cell, 126:663-676 (2006)).They successfully reprogrammed mouse embryonic fibroblasts (MEFs) andadult fibroblasts to pluripotent ES-like cells after viral-mediatedtransduction of the four transcription factors Oct4, Sox2, c-myc, andKlf4 followed by selection for activation of the Oct4 target gene Fbx15(FIG. 2A). Cells that had activated Fbx15 were coined iPS (inducedpluripotent stem) cells and were shown to be pluripotent by theirability to form teratomas, although the were unable to generate livechimeras. This pluripotent state was dependent on the continuous viralexpression of the transduced Oct4 and Sox2 genes, whereas the endogenousOct4 and Nanog genes were either not expressed or were expressed at alower level than in ES cells, and their respective promoters were foundto be largely methylated. This is consistent with the conclusion thatthe Fbx15-iPS cells did not correspond to ES cells but may haverepresented an incomplete state of reprogramming. While geneticexperiments had established that Oct4 and Sox2 are essential forpluripotency (Chambers and Smith, Oncogene, 23:7150-7160 (2004); Ivanonaet al., Nature, 442:5330538 (2006); Masui et al., Nat Cell Biol,9:625-635 (2007)), the role of the two oncogenes c-myc and Klf4 inreprogramming is less clear. Some of these oncogenes may, in fact, bedispensable for reprogramming, as both mouse and human iPS cells havebeen obtained in the absence of c-myc transduction, although with lowefficiency (Nakagawa et al., Nat Biotechnol, 26:191-106 (2008); Werninget al., Nature, 448:318-324 (2008); Yu et al., Science, 318: 1917-1920(2007)).

MAPC

MAPC is an acronym for “multipotent adult progenitor cell” (non-ES,non-EG, non-germ). Genes found in ES cells also have been found in MAPCs(e.g., telomerase, Oct 3/4, rex-1, rox-1, sox-2). Oct 3/4 (Oct 3A inhumans) appears to be specific for ES and germ cells. MAPC represents amore primitive progenitor cell population than MSC and demonstratesdifferentiation capability encompassing the epithelial, endothelial,neural, myogenic, hematopoietic, osteogenic, hepatogenic, chondrogenicand adipogenic lineages (Verfailie, C. M., Trends Cell Biol, 12:502-8,2002, Jahagirdar, B. N., et al., Exp Hematol, 29:543-56, 2001; Reyes, M.and C. M. Verfaillie, Ann N Y Acad Sci, 938:231-233, 2001; Jiang, Y. etal., Exp Hematol, 30896-904, 2002; and Jiang, Y. et al., Nature,418:41-9, 2002).

Human MAPCs are described in U.S. Pat. No. 7,015,037 and U.S.application Ser. No. 10/467,963. MAPCs have been identified in othermammals. Murine MAPCs, for example, also are described in U.S. Pat. No.7,015,037 and U.S. application Ser. No. 10/467,963. Rat MAPCs also aredescribed in U.S. application Ser. No. 10/467,963.

Isolation and Growth of MAPCs

Methods of MAPC isolation are known in the art. See, for example, U.S.Pat. No. 7,015,037 and U.S. application Ser. No. 10/467,963, and themethods contained therein, along with the characterization (phenotype)of MAPCs, which are incorporated herein by reference.

MAPCs were initially isolated from bone marrow, but subsequently wereestablished from other tissues, including brain and muscle (Jiang, Y. etal., Exp Hematol, 30896-904, 2002). Thus, MAPCs can be isolated frommultiple sources, including bone marrow, placenta, umbilical cord andcord blood, muscle, brain, liver, spinal cord, blood and skin. Forexample, MAPCs can be derived from bone marrow aspirates, which can beobtained by standard means available to those of skill in the art (see,for example, Muschler, G. F., et al., 1997; Batinic, D., et al., 1990).It, therefore, now is possible for those skilled in the art to obtainbone marrow aspirates, brain or liver biopsies, and other organs, and toisolate the cells using positive or negative selection techniquesavailable to those skilled in the art, relying upon the genes that areexpressed (or not expressed) in these cells (e.g., by functional ormorphological assays such as those disclosed in the above-referencedapplications, which have been incorporated herein by reference).

MAPCs from Human Bone Marrow

MAPCs do not express the common leukocyte antigen CD45 or erythroblastspecific glycophorin-A (Gly-A). As described in U.S. Pat. No. 7,015,037,which is incorporated by reference herein for the methods disclosedtherein, a mixed population of cells was subjected to a Ficoll Hypaqueseparation. The cells then were subjected to negative selection usinganti-CD45 and anti-Gly-A antibodies, depleting the population of CD45⁺and Gly-A⁺ cells, and the remaining approximately 0.1% of marrowmononuclear cells then were recovered. Cells also could be plated infibronectin-coated wells and cultured as described below for 2-4 weeksto deplete the cells of CD45⁺ and Gly-A⁺ cells. In cultures of adherentbone marrow cells, many adherent stromal cells undergo replicativesenescence around cell doubling 30 and a more homogenous population ofcells continues to expand and maintains long telomeres.

Alternatively, positive selection could be used to isolate cells via acombination of cell-specific markers. Both positive and negativeselection techniques are available to those of skill in the art, andnumerous monoclonal and polyclonal antibodies suitable for negativeselection purposes are also available in the art (see, for example,Leukocyte Typing V, Schlossman, et al., Eds., 1995, Oxford UniversityPress) and are commercially available from a number of sources.

Techniques for mammalian cell separation from a mixture of cellpopulations also have been described by Schwartz, et al. in U.S. Pat.No. 5,759,793 (magnetic separation), Basch et al., 1983 (immunoaffinitychromatography), and Wysocki and Sato, 1978 (fluorescence-activated cellsorting).

Culturing MAPC

MAPCs isolated as described herein can be cultured using methodsdisclosed herein and in U.S. Pat. No. 7,015,037, which is incorporatedby reference herein for these methods.

Additional Culture Methods

In additional experiments, the density at which MAPCs are cultured canvary from about 100 cells/cm² to about 150 cells/cm² to about 10,000cells/cm², including about 200 cells/cm² to about 1500 cells/cm² toabout 2000 cells/cm². The density can vary between species.Additionally, optimal density can vary depending on culture conditionsand source of cells. It is within the skill of the ordinary artisan todetermine the optimal density for a given set of culture conditions andcells.

Also, effective atmospheric oxygen concentrations of less than about10%, including about 3-5%, can be used at any time during the isolation,growth and differentiation of MAPCs in culture.

In an embodiment specific for MAPCs, supplements are cellular factors orcomponents that allow MAPCs to retain the ability to differentiate intoall three lineages. This may be indicated by the expression of specificmarkers of the undifferentiated state. MAPCs, for example,constitutively express Oct 3/4 (Oct 3A) and maintain high levels oftelomerase. Assays for monitoring gene expression are well known in theart (e.g., RT-PCR) and can be conducted using standard methodology.

Cell Culture

In general, cells useful for the invention can be maintained andexpanded in culture medium that is available to and well-known in theart. Such media include, but are not limited to, Dulbecco's ModifiedEagle's Medium® (DMEM), DMEM F12 Medium®, Eagle's Minimum EssentialMedium®, F-12K Medium®, Iscove's Modified Dulbecco's Medium® andRPMI-1640 Medium®. Many media are also available as low-glucoseformulations, with or without sodium pyruvate.

Also contemplated in the present invention is supplementation of cellculture medium with mammalian sera. Sera often contain cellular factorsand components that are necessary for viability and expansion. Examplesof sera include fetal bovine serum (FBS), bovine serum (BS), calf serum(CS), fetal calf serum (FCS), newborn calf serum (NCS), goat serum (GS),horse serum (HS), human serum, chicken serum, porcine serum, sheepserum, rabbit serum, serum replacements and bovine embryonic fluid. Itis understood that sera can be heat-inactivated at 55-65° C. if deemednecessary to inactivate components of the complement cascade.

Additional supplements also can be used advantageously to supply thecells with the necessary trace elements for optimal growth andexpansion. Such supplements include insulin, transferrin, sodiumselenium and combinations thereof. These components can be included in asalt solution such as, but not limited to, Hanks' Balanced SaltSolution® (HBSS), Earle's Salt Solution®, antioxidant supplements,MCDB-201® supplements, phosphate buffered saline (PBS), ascorbic acidand ascorbic acid-2-phosphate, as well as additional amino acids. Manycell culture media already contain amino acids, however, some requiresupplementation prior to culturing cells. Such amino acids include, butare not limited to, L-alanine, L-arginine, L-aspartic acid,L-asparagine, L-cysteine, L-cystine, L-glutamic acid, L-glutamine,L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine,L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan,L-tyrosine, and L-valine. It is well within the skill of one in the artto determine the proper concentrations of these supplements.

Hormones also can be advantageously used in the cell cultures of thepresent invention and include, but are not limited to, D-aldosterone,diethylstilbestrol (DES), dexamethasone, β-estradiol, hydrocortisone,insulin, prolactin, progesterone, somatostatin/human growth hormone(HGH), thyrotropin, thyroxine and L-thyronine.

Lipids and lipid carriers also can be used to supplement cell culturemedia, depending on the type of cell and the fate of the differentiatedcell. Such lipids and carriers can include, but are not limited to,cyclodextrin (α, β, γ), cholesterol, linoleic acid conjugated toalbumin, linoleic acid and oleic acid conjugated to albumin,unconjugated linoleic acid, linoleic-oleic-arachidonic acid conjugatedto albumin and oleic acid unconjugated and conjugated to albumin, amongothers.

Also contemplated in the present invention is the use of feeder celllayers. Feeder cells are used to support the growth of fastidiouscultured cells such as ES cells. Feeder cells are normal cells that havebeen inactivated by γ-irradiation. In culture, the feeder layer servesas a basal layer for other cells and supplies cellular factors withoutfurther growth or division of their own (Lim, J. W. and Bodnar, A.,2002). Examples of feeder layer cells are typically human diploid lungcells, mouse embryonic fibroblasts and Swiss mouse embryonicfibroblasts, but can be any post-mitotic cell that is capable ofsupplying cellular components and factors that are advantageous inallowing optimal growth, viability and expansion of stem cells. In manycases, feeder cell layers are not necessary to keep ES cells in anundifferentiated, proliferative state, as leukemia inhibitory factor(LIF) has anti-differentiation properties. Therefore, supplementationwith LIF can be used to maintain MAPC in some species in anundifferentiated state.

Cells may be cultured in low-serum or serum-free culture medium.Serum-free medium used to culture MAPCs is described in U.S. Pat. No.7,015,037. Many cells have been grown in serum-free or low-serum medium.In this case, the medium is supplemented with one or more growthfactors. Commonly used growth factors include, but are not limited to,bone morphogenic protein, basis fibroblast growth factor,platelet-derived growth factor and epidermal growth factor. See, forexample, U.S. Pat. Nos. 7,169,610; 7,109,032; 7,037,721; 6,617,161;6,617,159; 6,372,210; 6,224,860; 6,037,174; 5,908,782; 5,766,951;5,397,706; and 4,657,866; all incorporated by reference herein forteaching growing cells in serum-free medium.

Cells in culture can be maintained either in suspension or attached to asolid support, such as extracellular matrix components. Stem cells oftenrequire additional factors that encourage their attachment to a solidsupport, such as type I and type II collagen, chondroitin sulfate,fibronectin, “superfibronectin” and fibronectin-like polymers, gelatin,poly-D and poly-L-lysine, thrombospondin and vitronectin. One embodimentof the present invention utilizes fibronectin. See, for example, Ohashiet al., Nature Medicine, 13:880-885 (2007); Matsumoto et al., JBioscience and Bioengineering, 105:350-354 (2008); Kirouac et al., CellStem Cell, 3:369-381(2008); Chua et al., Biomaterials, 26:2537-2547(2005); Drobinskaya et al., Stem Cells, 26:2245-2256 (2008);Dvir-Ginzberg et al., FASEB J, 22:1440-1449 (2008); Turner et al., JBiomed Mater Res Part B: Appl Biomater, 82B:156-168 (2007); and Miyazawaet al., Journal of Gastroenterology and Hepatology, 22:1959-1964(2007)).

Cells may also be grown in “3D” (aggregated) cultures. An example isU.S. Provisional Patent Application No. 61/022,121, filed Jan. 18, 2008.

Once established in culture, cells can be used fresh or frozen andstored as frozen stocks, using, for example, DMEM with 40% FCS and 10%DMSO. Other methods for preparing frozen stocks for cultured cells alsoare available to those skilled in the art.

Methods of identifying and subsequently separating differentiated cellsfrom their undifferentiated counterparts can be carried out by methodswell known in the art. Cells that have been induced to differentiateusing methods of the present invention can be identified by selectivelyculturing cells under conditions whereby differentiated cells outnumberundifferentiated cells. Similarly, differentiated cells can beidentified by morphological changes and characteristics that are notpresent on their undifferentiated counterparts, such as cell size andthe complexity of intracellular organelle distribution. Alsocontemplated are methods of identifying differentiated cells by theirexpression of specific cell-surface markers such as cellular receptorsand transmembrane proteins. Monoclonal antibodies against thesecell-surface markers can be used to identify differentiated cells.Detection of these cells can be achieved through fluorescence activatedcell sorting (FACS) and enzyme-linked immunosorbent assay (ELISA). Fromthe standpoint of transcriptional upregulation of specific genes,differentiated cells often display levels of gene expression that aredifferent from undifferentiated cells. Reverse-transcription polymerasechain reaction, or RT-PCR, also can be used to monitor changes in geneexpression in response to differentiation. Whole genome analysis usingmicroarray technology also can be used to identify differentiated cells.

Accordingly, once differentiated cells are identified, they can beseparated from their undifferentiated counterparts, if necessary. Themethods of identification detailed above also provide methods ofseparation, such as FACS, preferential cell culture methods, ELISA,magnetic beads and combinations thereof. One embodiment of the presentinvention comtemplates the use of FACS to identify and separate cellsbased on cell-surface antigen expression.

Pharmaceutical Formulations

Any of the cells produced by the methods described herein can be used inthe clinic to treat a subject. They can, therefore, be formulated into apharmaceutical composition. Therefore, in certain embodiments, theisolated or purified cell populations are present within a compositionadapted for and suitable for delivery, i.e., physiologically compatible.Accordingly, compositions of the cell populations will often furthercomprise one or more buffers (e.g., neutral buffered saline or phosphatebuffered saline), carbohydrates (e.g., glucose, mannose, sucrose ordextrans), mannitol, proteins, polypeptides or amino acids such asglycine, antioxidants, bacteriostats, chelating agents such as EDTA orglutathione, adjuvants (e.g., aluminum hydroxide), solutes that renderthe formulation isotonic, hypotonic or weakly hypertonic with the bloodof a recipient, suspending agents, thickening agents and/orpreservatives.

In other embodiments, the isolated or purified cell populations arepresent within a composition adapted for or suitable for freezing orstorage.

In many embodiments the purity of the cells for administration to asubject is about 100%. In other embodiments it is 95% to 100%. In someembodiments it is 85% to 95%. Particularly in the case of admixtureswith other cells, the percentage can be about 10%-15%, 15%-20%, 20%-25%,25%-30%, 30%-35%, 35%-40%, 40%-45%, 45%-50%, 60%-70%, 70%-80%, 80%-90%,or 90%-95%. Or isolation/purity can be expressed in terms of celldoublings where the cells have undergone, for example, 10-20, 20-30,30-40, 40-50 or more cell doublings.

The numbers of cells in a given volume can be determined by well knownand routine procedures and instrumentation. The percentage of the cellsin a given volume of a mixture of cells can be determined by much thesame procedures. Cells can be readily counted manually or by using anautomatic cell counter. Specific cells can be determined in a givenvolume using specific staining and visual examination and by automatedmethods using specific binding reagent, typically antibodies,fluorescent tags, and a fluorescence activated cell sorter.

The choice of formulation for administering the cells for a givenapplication will depend on a variety of factors. Prominent among thesewill be the species of subject, the nature of the disorder, dysfunction,or disease being treated and its state and distribution in the subject,the nature of other therapies and agents that are being administered,the optimum route for administration, survivability via the route, thedosing regimen, and other factors that will be apparent to those skilledin the art. In particular, for instance, the choice of suitable carriersand other additives will depend on the exact route of administration andthe nature of the particular dosage form.

For example, cell survival can be an important determinant of theefficacy of cell-based therapies. This is true for both primary andadjunctive therapies. Another concern arises when target sites areinhospitable to cell seeding and cell growth. This may impede access tothe site and/or engraftment there of therapeutic cells. Variousembodiments of the invention comprise measures to increase cell survivaland/or to overcome problems posed by barriers to seeding and/or growth.

Final formulations of the aqueous suspension of cells/medium willtypically involve adjusting the ionic strength of the suspension toisotonicity (i.e., about 0.1 to 0.2) and to physiological pH (i.e.,about pH 6.8 to 7.5). The final formulation will also typically containa fluid lubricant, such as maltose, which must be tolerated by the body.Exemplary lubricant components include glycerol, glycogen, maltose andthe like. Organic polymer base materials, such as polyethylene glycoland hyaluronic acid as well as non-fibrillar collagen, preferablysuccinylated collagen, can also act as lubricants. Such lubricants aregenerally used to improve the injectability, intrudability anddispersion of the injected biomaterial at the site of injection and todecrease the amount of spiking by modifying the viscosity of thecompositions. This final formulation is by definition the cells in apharmaceutically acceptable carrier.

The cells are subsequently placed in a syringe or other injectionapparatus for precise placement at the site of the tissue defect. Theterm “injectable” means the formulation can be dispensed from syringeshaving a gauge as low as 25 under normal conditions under normalpressure without substantial spiking. Spiking can cause the compositionto ooze from the syringe rather than be injected into the tissue. Forthis precise placement, needles as fine as 27 gauge (200 μI.D.) or even30 gauge (150 μI.D.) are desirable. The maximum particle size that canbe extruded through such needles will be a complex function of at leastthe following: particle maximum dimension, particle aspect ratio(length:width), particle rigidity, surface roughness of particles andrelated factors affecting particle:particle adhesion, the viscoelasticproperties of the suspending fluid, and the rate of flow through theneedle. Rigid spherical beads suspended in a Newtonian fluid representthe simplest case, while fibrous or branched particles in a viscoelastic fluid are likely to be more complex.

The desired isotonicity of the compositions of this invention may beaccomplished using sodium chloride, or other pharmaceutically acceptableagents such as dextrose, boric acid, sodium tartrate, propylene glycol,or other inorganic or organic solutes. Sodium chloride is preferredparticularly for buffers containing sodium ions.

Viscosity of the compositions, if desired, can be maintained at theselected level using a pharmaceutically acceptable thickening agent.Methylcellulose is preferred because it is readily and economicallyavailable and is easy to work with. Other suitable thickening agentsinclude, for example, xanthan gum, carboxymethyl cellulose,hydroxypropyl cellulose, carbomer, and the like. The preferredconcentration of the thickener will depend upon the agent selected. Theimportant point is to use an amount, which will achieve the selectedviscosity. Viscous compositions are normally prepared from solutions bythe addition of such thickening agents.

A pharmaceutically acceptable preservative or stabilizer can be employedto increase the life of cell/medium compositions. If such preservativesare included, it is well within the purview of the skilled artisan toselect compositions that will not affect the viability or efficacy ofthe cells.

Those skilled in the art will recognize that the components of thecompositions should be chemically inert. This will present no problem tothose skilled in chemical and pharmaceutical principles. Problems can bereadily avoided by reference to standard texts or by simple experiments(not involving undue experimentation) using information provided by thedisclosure, the documents cited herein, and generally available in theart.

Sterile injectable solutions can be prepared by incorporating thecells/medium utilized in practicing the present invention in therequired amount of the appropriate solvent with various amounts of theother ingredients, as desired.

In some embodiments, cells/medium are formulated in a unit dosageinjectable form, such as a solution, suspension, or emulsion.Pharmaceutical formulations suitable for injection of cells/mediumtypically are sterile aqueous solutions and dispersions. Carriers forinjectable formulations can be a solvent or dispersing mediumcontaining, for example, water, saline, phosphate buffered saline,polyol (for example, glycerol, propylene glycol, liquid polyethyleneglycol, and the like), and suitable mixtures thereof.

The skilled artisan can readily determine the amount of cells andoptional additives, vehicles, and/or carrier in compositions to beadministered in methods of the invention. Typically, any additives (inaddition to the cells) are present in an amount of 0.001 to 50 wt % insolution, such as in phosphate buffered saline. The active ingredient ispresent in the order of micrograms to milligrams, such as about 0.0001to about 5 wt %, preferably about 0.0001 to about 1 wt %, mostpreferably about 0.0001 to about 0.05 wt % or about 0.001 to about 20 wt%, preferably about 0.01 to about 10 wt %, and most preferably about0.05 to about 5 wt %.

In some embodiments cells are encapsulated for administration,particularly where encapsulation enhances the effectiveness of thetherapy, or provides advantages in handling and/or shelf life.Encapsulation in some embodiments where it increases the efficacy ofcell mediated immunosuppression may, as a result, also reduce the needfor immunosuppressive drug therapy.

Also, encapsulation in some embodiments provides a barrier to asubject's immune system that may further reduce a subject's immuneresponse to the cells (which generally are not immunogenic or are onlyweakly immunogenic in allogeneic transplants), thereby reducing anygraft rejection or inflammation that might occur upon administration ofthe cells.

Cells may be encapsulated by membranes, as well as capsules, prior toimplantation. It is contemplated that any of the many methods of cellencapsulation available may be employed. In some embodiments, cells areindividually encapsulated. In some embodiments, many cells areencapsulated within the same membrane. In embodiments in which the cellsare to be removed following implantation, a relatively large sizestructure encapsulating many cells, such as within a single membrane,may provide a convenient means for retrieval.

A wide variety of materials may be used in various embodiments formicroencapsulation of cells. Such materials include, for example,polymer capsules, alginate-poly-L-lysine-alginate microcapsules, bariumpoly-L-lysine alginate capsules, barium alginate capsules,polyacrylonitrile/polyvinylchloride (PAN/PVC) hollow fibers, andpolyethersulfone (PES) hollow fibers.

Techniques for microencapsulation of cells that may be used foradministration of cells are known to those of skill in the art and aredescribed, for example, in Chang, P., et al., 1999; Matthew, H. W., etal., 1991; Yanagi, K., et al., 1989; Cai Z. H., et al., 1988; Chang, T.M., 1992 and in U.S. Pat. No. 5,639,275 (which, for example, describes abiocompatible capsule for long-term maintenance of cells that stablyexpress biologically active molecules. Additional methods ofencapsulation are in European Patent Publication No. 301,777 and U.S.Pat. Nos. 4,353,888; 4,744,933; 4,749,620; 4,814,274; 5,084,350;5,089,272; 5,578,442; 5,639,275; and 5,676,943. All of the foregoing areincorporated herein by reference in parts pertinent to encapsulation ofcells.

Certain embodiments incorporate cells into a polymer, such as abiopolymer or synthetic polymer. Examples of biopolymers include, butare not limited to, fibronectin, fibin, fibrinogen, thrombin, collagen,and proteoglycans. Other factors, such as the cytokines discussed above,can also be incorporated into the polymer. In other embodiments of theinvention, cells may be incorporated in the interstices of athree-dimensional gel. A large polymer or gel, typically, will besurgically implanted. A polymer or gel that can be formulated in smallenough particles or fibers can be administered by other common, moreconvenient, non-surgical routes.

In the case of treating liver deficiency, in particular, the cells maybe enclosed in a device that can be implanted in a subject. Cells can beimplanted in or near the liver or elsewhere to replace or supplementliver function. Cells can also be implanted without being in a device,e.g., in existing liver tissue.

Dosing

Compositions can be administered in dosages and by techniques well knownto those skilled in the medical and veterinary arts taking intoconsideration such factors as the age, sex, weight, and condition of theparticular patient, and the formulation that will be administered (e.g.,solid vs. liquid). Doses for humans or other mammals can be determinedwithout undue experimentation by the skilled artisan, from thisdisclosure, the documents cited herein, and the knowledge in the art.

The dose of cells/medium appropriate to be used in accordance withvarious embodiments of the invention will depend on numerous factors. Itmay vary considerably for different circumstances. The parameters thatwill determine optimal doses to be administered for primary andadjunctive therapy generally will include some or all of the following:the disease being treated and its stage; the species of the subject,their health, gender, age, weight, and metabolic rate; the subject'simmunocompetence; other therapies being administered; and expectedpotential complications from the subject's history or genotype. Theparameters may also include: whether the cells are syngeneic,autologous, allogeneic, or xenogeneic; their potency (specificactivity); the site and/or distribution that must be targeted for thecells/medium to be effective; and such characteristics of the site suchas accessibility to cells/medium and/or engraftment of cells. Additionalparameters include co-administration with other factors (such as growthfactors and cytokines). The optimal dose in a given situation also willtake into consideration the way in which the cells/medium areformulated, the way they are administered, and the degree to which thecells/medium will be localized at the target sites followingadministration. Finally, the determination of optimal dosing necessarilywill provide an effective dose that is neither below the threshold ofmaximal beneficial effect nor above the threshold where the deleteriouseffects associated with the dose outweighs the advantages of theincreased dose.

The optimal dose of cells for some embodiments will be in the range ofdoses used for autologous, mononuclear bone marrow transplantation. Forfairly pure preparations of cells, optimal doses in various embodimentswill range from 10⁴ to 10⁸ cells/kg of recipient mass peradministration. In some embodiments the optimal dose per administrationwill be between 10⁵ to 10⁷ cells/kg. In many embodiments the optimaldose per administration will be 5×10⁵ to 5×10⁶ cells/kg. By way ofreference, higher doses in the foregoing are analogous to the doses ofnucleated cells used in autologous mononuclear bone marrowtransplantation. Some of the lower doses are analogous to the number ofCD34⁺ cells/kg used in autologous mononuclear bone marrowtransplantation.

It is to be appreciated that a single dose may be delivered all at once,fractionally, or continuously over a period of time. The entire dosealso may be delivered to a single location or spread fractionally overseveral locations.

In various embodiments, cells/medium may be administered in an initialdose, and thereafter maintained by further administration. Cells/mediummay be administered by one method initially, and thereafter administeredby the same method or one or more different methods. The levels can bemaintained by the ongoing administration of the cells/medium. Variousembodiments administer the cells/medium either initially or to maintaintheir level in the subject or both by intravenous injection. In avariety of embodiments, other forms of administration, are used,dependent upon the patient's condition and other factors, discussedelsewhere herein.

It is noted that human subjects are treated generally longer thanexperimental animals; but, treatment generally has a length proportionalto the length of the disease process and the effectiveness of thetreatment. Those skilled in the art will take this into account in usingthe results of other procedures carried out in humans and/or in animals,such as rats, mice, non-human primates, and the like, to determineappropriate doses for humans. Such determinations, based on theseconsiderations and taking into account guidance provided by the presentdisclosure and the prior art will enable the skilled artisan to do sowithout undue experimentation.

Suitable regimens for initial administration and further doses or forsequential administrations may all be the same or may be variable.Appropriate regimens can be ascertained by the skilled artisan, fromthis disclosure, the documents cited herein, and the knowledge in theart.

The dose, frequency, and duration of treatment will depend on manyfactors, including the nature of the disease, the subject, and othertherapies that may be administered. Accordingly, a wide variety ofregimens may be used to administer the cells/medium.

In some embodiments cells/medium are administered to a subject in onedose. In others cells/medium are administered to a subject in a seriesof two or more doses in succession. In some other embodiments whereincells/medium are administered in a single dose, in two doses, and/ormore than two doses, the doses may be the same or different, and theyare administered with equal or with unequal intervals between them.

Cells/medium may be administered in many frequencies over a wide rangeof times. In some embodiments, they are administered over a period ofless than one day. In other embodiment they are administered over two,three, four, five, or six days. In some embodiments they areadministered one or more times per week, over a period of weeks. Inother embodiments they are administered over a period of weeks for oneto several months. In various embodiments they may be administered overa period of months. In others they may be administered over a period ofone or more years. Generally lengths of treatment will be proportionalto the length of the disease process, the effectiveness of the therapiesbeing applied, and the condition and response of the subject beingtreated.

Uses for the Cells

The liver plays a major role in metabolism, including plasma proteinsynthesis (e.g. albumin and coagulation factors), glycogen storage,decomposition of red blood cells, and detoxification. One of the majorimpediments to the use of hepatocytes in at least four areas of medicineis, in general, the scarcity of human hepatocytes.

(1) Therapy of Liver Failure with Hepatocyte Transplantation:

One of the unique features of the liver is its enormous naturalregeneration ability. This regeneration is due in large part to there-entry of terminally differentiated hepatocytes in the cell cycle,resulting in multiple cell divisions to regenerate the liver. When thehepatocytes are damaged, liver stem/progenitor cells, termed oval cellsin rodents, located in the peri-portal zone, are activated anddifferentiate to mature hepatocytes (ref). Hence, most liver diseasesending in liver failure are caused by a combination of decreasedproliferation of hepatocytes and exhaustion of the stem/progenitor cellpool. Liver failure is caused by a number of disorders, includingcirrhosis due to infections, excessive alcohol consumption, genetic andidiopatic reasons. In addition, acute liver failure is caused byingestion of certain drugs or foods. Liver transplantation is the onlysuccessful treatment for end stage liver disease, and is in manyinstances also the only curative therapy for certain forms of geneticdisorders of the liver. Many liver disorders treated by whole livertransplantation result from hepatocyte dysfunction. As a consequence,there has been great interest in hepatocyte transplantation for thetreatment of acute and chronic liver failure, as well as inheritedmetabolic disorders. There is significant evidence that graftedhepatocytes can assume the full range of liver functions in vivo.Hepatocyte transplantation has several advantages over whole livertransplant: lower morbidity, a single donor organ can be used forseveral recipients, cells can be cryopreserved, and cells grafts areless immunogenic than whole organ grafts. However, lack of donor cellscurtails further exploration of this therapy.

Accordingly the invention is also directed to methods of treating liverdeficiencies by administering the cells of the invention to a subjectwith the liver deficiency. Such deficiencies include, but are notlimited to, toxic liver disease, metabolic liver disease, acute livernecrosis, effects of acetaminophen, hemochromatosis, Wilson's Disease,Crigler Najar, hereditary tyrosinemia, familial intrahepatic cholestatistype 3, ornithine transcarbamylase (OTC) deficiency, and urea cycledisorder.

Further diseases include, but are not limited to viral hepatitis,chronic viral hepatitis A, B, C, acute hepatitis A, B, C, D, E,cytomegalovirus and herpes simplex virus; liver dysfunction in otherinfectious diseases such as, without limitation, toxoplasmosis,hepatosplenic schistosomiasis, liver disease in syphilis, leptospirosisand amoebiasis; metabolic diseases such as, without limitation,haemochromatosis, Gilbert's syndrome, Dubin-Johnson syndrome and Rotor'ssyndrome; alcoholic liver disease such as, without limitation, fattyliver, fibrosis, sclerosis and cirrhosis; and toxic liver disease.

(2) Bioartificial Liver (BAL) Devices

In patients with terminal liver failure, the use of bioartificial liverdevices has been proposed to bridge the time to liver transplantation(ref). BAL devices are designed to support the detoxification functionsperformed by the liver, hence decreasing the risk and severity of CNScomplications associated with acute liver failure. BAL devices couldbenefit three groups of patients; those with fulminant hepatic failure,those waiting for an imminent transplant, and those with early failureof a liver transplant. Although some positive results have been seen inpatients with liver failure, further exploration of the usefulness ofBAL devices has been hampered by lack of suitable cells. Currently,tumor-derived cell lines or animal cells, which might be associated withpossible tumor cell seeding, immune responses, and xeno-zoonoses, areused. The availability of cells with full mature hepatic function ofhuman origin, would enable investigators to further test and optimizeBAL devices to bridge patients till the liver spontaneously regeneratesor a donor-liver is available. Although clinical trials have in generalnot been successful, some encouraging results have been seen in patientswith acute liver failure. Accordingly, the cells of the invention can beused in such bioartificial liver devices.

(3) Pharmaceutical Testing

Pharmaceutical testing is moving more and more from in vivoexperimentation to in vitro studies. Over the past decade, in vitromodels were established, such as precision-cut liver slices, primaryhepatocytes, and liver cell lines. A few studies examining the relevanceof drug testing with hepatocyte cell lines, found that cell lines poorlyreproduce and predict drug metabolism and hepatotoxicity as opposed toprimary hepatocytes or liver slices. Thus, primary human hepatocytes arethe “gold standard” for in vitro drug testing. However, the limitedsupply of human hepatocytes and the fact that such hepatocytes may notrepresent the genetic variation in society, limit the possibility ofdetecting potential drug toxicities. Consequently, development of stemcells from a diverse group of donors (for instance by generating IPScells) and differentiation to hepatocytes with differing cytochrome P450profiles would allow drug testing to more closely examine and predictpotential problems for particular groups or individuals.

Accordingly, the cells of the invention can be used in such testingmethods.

(4) Study of and Drug Development Against Human Hepatitis Viruses

A final area where generating hepatocytes from stem cells would aidhuman health significantly is in the area of viral hepatitis. Most invitro studies on the hepatitis viruses have involved primary humanhepatocytes. However, again, the limited availability of human tissueand also subject to the variability of the source material impedes thesetypes of studies.

Accordingly, the cells of the invention can be used in such studies andin drug development.

The present invention is additionally described by way of the followingillustrative, non-limiting Example that provides a better understandingof the present invention and of its many advantages.

EXAMPLES Example 1

I. Methodology

1. Rat MAPC Culture

A rat MAPC cell line was derived from bone marrow of a male Fischer(F344) rat by Prof. Y. Jiang at the University of Minnesota,Minneapolis, USA. This established rat MAPC line was used in theexperiments described below. Undifferentiated rat MAPCs were cultured atlow cell density (˜300 cells/cm²) in low serum (2%) and oxygen (5%)conditions. Expansion medium consisted of DMEM (60%), MCDB (40%)supplemented with leukemia inhibiting factor (LIF), growth factors (10ng/ml PDGF and 10 ng/ml EGF), dexamethasone (5×10⁻⁸ M), penicillin andstreptomycin (1×), ascorbic acid (1×), beta-mercaptoethanol andinsulin-transferrin-selenium with linoleic acid (ITS+1). Culture dishes(˜58 cm²) were coated with rat fibronectin for 30 minutes at 37° C. orfor 60 minutes at room temperature. In order to inhibit initiation ofdifferentiation, intercellular contact was prevented. Therefore, every48 hours, MAPC were trypsinized (0.25 or 0.05% trypsin), centrifuged at2100 RPM for 6.5 minutes, counted and replated at ˜300 cells/cm².

2. Quality Control of Undifferentiated Rat MAPCs

On a regular basis, cytogenetics, Oct4 level (mRNA and staining) andphenotype of the undifferentiated cells were determined and theirmultilineage capacity was tested.

3. In Vitro Differentiation of MAPCs

3.1 Initiation of In Vitro Hepatic Differentiation

Undifferentiated MAPCs were expanded at large scale until severalmillion cells were obtained. Cells then were plated at 50,000-60,000cells/cm² in Matrigel (2%) coated wells. Initially, cells were culturedin expansion medium until they reached 80-90% confluency 16 hours later.Then, cells were washed twice with PBS and the medium was switched todifferentiation medium. Basal differentiation medium consisted of DMEM(60%), MCDB (40%), ascorbic acid (1×), penicillin/streptomycin (1×),beta-mercaptoethanol, ITS (0.25×), LA-BSA (0.25×) and dexamethasone(10⁻⁶M).

3.2 Choice of Recombinant Cytokines and Medium Components

Understanding the molecular signals favoring liver development isessential for the development of a logical in vitro differentiationprotocol to modulate stem cell differentiation towards the hepatic fate.With this in mind, different cytokines at variable concentrations andchronological orders were investigated for their hepatocyte inducingcapacity in rMAPCs. Cytokines tested included Nodal, its co-receptorCripto, ActivinA, Wnt3a, BMP4, bFGF, aFGF, FGF8b, EGF, Nicotinamide,heparan sulphate proteoglycan, Oncostatin M, HGF and Follistatin. Allcytokines were purchased from R&D, Minneapolis, USA. Apart from thecytokines, medium components also play an important role in guidingcells towards a specific fate. The effect of changing the percentage ofserum, concentration of dexamethasone (none, 5×10⁻⁸ M and 10⁻⁶ M) andinsulin was tested. Control samples consisted of rMAPCs without theaddition of cytokines according to the standard protocol.

4. qRT-PCR

Total RNA was extracted using the Qiagen mini-kit. Next, samples weretreated with Turbo DNAse free (Ambion, USA) for 30 minutes at 37° C. Oneμg of RNA was used for each cDNA-synthesis using Superscript III reversetranscriptase (Invitrogen, USA). Semiqualitative PCR was performed withSybr Green (Invitrogen, USA) on the Eppfendorf realtime PCR machine (40cycles). As controls, fetal (ED15), mature total liver, spleen oruniversal RNA were used, depending upon the markers tested. GAPDH wasapplied as a house-keeping gene. Results were shown in delta CT=CT(marker of interest)−CT (GAPDH) and were compared to the delta CT of theundifferentiated cells and the positive control. The lower the delta CT,the higher the expression of that gene.

5. Immunohistochemistry

Cultured cells were fixed with 4% paraformaldehyde for 10 minutes atroom temperature. The following antibodies were used: Dako Rabbitanti-Albumin (A0001, 1:2000 dilution), ICN Rabbit anti-Albumin (55711,1:10000 dilution), Chemicon Mouse anti-CK18 (CBL177, 1:10 dilution),Santa Cruz Rabbit anti-HNF1 (sc-8986, 1:50 dilution), NeoMarkers Rabbitanti-alpha-fetoprotein (RB-365A, 1:300 dilution) and Chemicon Rabbitanti-von Willebrandt factor (AB7356, 1:200). After incubation with theprimary antibodies, samples were labeled with EnVision+Horseradishperoxidase (K4002 anti-Rabbit or K4000 anti-Mouse). Staining wascompleted with an incubation of DAB substrate-chromogen. Antibodies weretitrated using primary rat hepatocytes or paraffin embedded normal ratliver. Negative controls included isotype controls used at the sameconcentration as the primary antibodies.

6. Funtional Assays

a. Glycogen staining. Cells were fixed with methanol at −20° C. for 10minutes and periodic acid staining was performed using a Sigma kit.

b. Albumin was measured in the medium every 48 hours beginning at day 10of differentiation, using competitive ELISA Protocol with rat and mousespecific antibodies, following the manufacturers instructions (Bethyl).

c. Capacity to conjugate bilirubin was investigated by addingunconjugated bilirubin-BSA (50 μM-100 μM) to the medium. After differenttime points (0-24 hours), medium was collected to measure bilirubinmono- and di-glucuronide with high performance liquid chromatography(HPLC).

d. Electron Microscopy. A minimum of 3 million cells were collected byscraping of the cells with collagenase or dispase, centrifuged and fixedin electron microscopy-specific fixing medium. Samples were furtheranalyzed by Prof. R. Devos, KU Leuven.

e. LDL-uptake (Biomedical Technologies, Inc). Cells were preincubatedwith fresh DMEM for 1 hour. Dil-Ac-LDL at a final concentration of 10μg/ml was added to the cells for 1 to 4 hours at 37° C. Afterwards,cells were washed three times with PBS and fixed to visualize cellularLDL uptake with immunofluorescence.

f. Urea Assay. Cells were incubated with ammonium chloride (2.5 to 5 μM)for 4-6 hours and urea concentrations were screened by a colorimetricassay (Gentaur).

g. Lectin staining. Cells were fixed with 4% para-formaldehyde andblocked with TBS/1% BSA for 1 hour, incubated with lectin fromBandeiraea simplicifolia at 10 μg/ml (Sigma) and washed with TBS 3times. Then, streptavidine-PE (BD Pharmingen) was added for 45 minutes.

h. CYP3A4 activity by testosterone hydroxylation. Cells were incubatedin differentiation medium containing 25 μM rifampcin for 48 hours andthe CYP3A4 substrate testosterone was added to a final concentration of100 μM in the presence or absence of 10 μM ketoconozole, a CYP3A4inhibitor. After a further 24 hours culture, the medium was collectedand CYP3A4 activity was measured using HPLC for the production of 6β-OHtestosterone. For all of the experiments described, mature rathepatocytes or rat liver tissue served as a positive control.

II. Results

1. Characterization of Undifferentiated rat MAPCs

One subclone of rat MAPCs expresses high levels of the pluripotentmarker Oct4 both at the mRNA and protein level. In addition, amicro-array analysis of this subclone showed the expression of Sal14. Incontrast to embryonic stem cells, however, no Nanog or Sox2 wasexpressed. Undifferentiated high Oct4-expressing MAPCs express earlymesendodermal markers, but no expression of mature hepatic markers suchas albumin, alphal-antitrypsin or tyrosine aminotransferase. FACSanalysis showed that rat MAPCs were positive for the surface marker CD31and negative for the mesenchymal marker CD44.

2. Differentiation of Rat MAPCs and Mouse ES Cells.

2.1 Choice of Cytokines and Medium Components

Among the large number of differentiation protocols (>200) tested, theoptimal protocol was selected based upon mRNA expression levels ofalbumin, alpha-fetoprotein, transthyretin, tyrosine aminotransferase andalpha-1 antitrypsine. In the final 21 day differentiation protocol, astandard protocol of four sequential combinations of cytokines wereused: (1) 50 ng/ml Wnt3a and 100 ng/ml ActivinA; (2) 10 ng/ml bFGF and50 ng/ml BMP4; (3) 50 ng/ml aFGF, 10 ng/ml FGF4 and 25 ng/ml FGF8b; and(4) 20 ng/ml HGF and 100 ng/ml Follistatin (FIG. 1). In order todiscriminate between hepatocyte- or biliary-like cells, Activin isinhibited by Follistatin. To verify whether the addition of thecytokines had a real hepatocyte inducing effect, differentiation wasperformed using basal differentiation medium only. A high concentrationof dexamethasone was used because some hepatocyte specific genes (i.e.,tyrosine aminotransferase, MRP2 and tryptophan 2,3 dioxygenase) areupregulated by glucocorticoids, as they contain a glucocorticoidresponse element. In the complete absence of serum, cell death occurred.However, using Wnt3a, differentiation was induced in serum-freeconditions. If no cytokines were added to the basal differentiatingmedium, 2% serum was added until day 12 and then stopped. Because highconcentrations of dexamethasone, together with insulin, can induceadipogenesis, a lower amount of insulin was used.

2.2 Morphology

Using light microscopy, an overview of the changes in morphology ofdifferentiating HO-rMAPC can be seen in FIG. 2. The top three panelsshow the changes in morphology after days 1, 2 and 3 of differentiation,respectively. The middle three panels show the changes in morphologyafter days 8, 12 and 14 of differentiation, respectively. The bottomthree panels show the changes in morphology after days 14, 18 and 20 ofdifferentiation, respectively. After days 12 and 14, endothelial-likecells can be seen. Clusters of rounded cells can be seen after days 14and 18. Cobble stone-epithelial like cells can be seen after day 18.After day 20, flattened, polygonal cells can be seen.

FIG. 3 shows, using light microscopy, the morphological changes observedduring differentiation of rMAPC-1 in differentiation medium withoutcytokine supplementation (control samples). The top three panels showthe morphological changes after days 1, 4 and 6 of differentiation. Thebottom three panels show the morphological changes after day 10, day 20at 40× and day 20 at 20×, respectively, of differentiation. Overall,cells appeared morphologically more homogeneous until day 20 compared tothe cytokine-containing differentiation cultures. Colonies of smallrounded cells also could be seen from day 15 on, similar to cellstreated with the standard protocol. Using the same cytokines, a similarheterogeneity is seen in differentiating mouse ES cells (data notshown).

Using electron microscopy, some cells showed characteristics of bothcholangiocyte-like and hepatocyte-like cells: bile duct-like structures(FIG. 4A-B); tonofilaments (FIG. 4C at arrow); polarized hepatocyte-likecells (FIG. 4D); metabolically active cells with much mitochondria FIG.4E at arrow); and tight junctions (FIG. 4F at arrow).

2.3 qRT-PCR Results on Differentiating Rat MAPCs

Results of the qRT-PCR analysis showed the expression of specific genesover time in two different clones of rMAPCs, as shown in Tables 1a and1b.

TABLE 1a rat clone 19 d 0 d 6 d 10 d 14 d 20 Oct4  5.9 +/− 1.0 (3) 14.8+/− 1.5 (3)  13.4 +/− 2.5 (3)  8.3 +/− 1.1 (3) 12.1 +/− 3.9 (3)  Mixl121.6 +/− 1.2 (3) 6.2 +/− 1.6 (3) 11.3 +/− 1.1 (3)  16.4 +/− 0.4 (3) 21.2 +/− 1.7 (3)  Eomes  8.5 +/− 0.2 (3) 4.5 +/− 0.2 (3) 8.0 +/− 0.5 (3)9.9 +/− 1.0 (3) 10.1 +/− 0.2 (3)  Brachyury 23.6 +/− 0.1 (3) 18.1 +/−3.0 (3)  18.8 +/− 1.1 (3)  14.7 +/− 0.8 (3)  19.2 +/− 2.0 (3)  Tm4sf221.7 +/− 1.8 (3) 3.2 +/− 0.4 (3) 6.4 +/− 1.1 (3) 7.5 +/− 0.4 (3) 9.1 +/−0.4 (3) Sox17  4.7 +/− 1.1 (2) 7.7 +/− 0.7 (2) 7.3 +/− 0.0 (2) 6.6 +/−0.7 (2) 6.7 +/− 1.5 (2) Foxa2  6.9 +/− 0.1 (2) 5.6 +/− 0.2 (2) 5.6 +/−0.3 (2) 5.5 +/− 0.3 (2) 7.0 +/− 0.1 (2) CXCR4 17.5 +/− 0.7 (3) 5.3 +/−1.3 (3) 8.6 +/− 0.8 (3) 9.8 +/− 0.7 (3) 8.6 +/− 1.6 (3) Flk1 19.5 +/−1.6 (3) 19.4 +/− 1.2 (3)  17.2 +/− 2.3 (3)  12.5 +/− 0.6 (3)  12.2 +/−0.9 (3)  E-cadherin 5.8 (1) 1.9 (1) 1.9 (1) 2.1 (1) 2.5 (1) Hex1  8.7+/− 0.0 (2) 4.2 +/− 0.5 (2) 5.4 +/− 0.1 (2) 5.8 +/− 0.1 (2) 6.9 +/− 0.3(2) Prox1  8.7 +/− 0.4 (3) 5.2 +/− 0.7 (3) 4.8 +/− 0.2 (3) 5.0 +/− 0.5(3) 5.4 +/− 0.4 (3) HNF1a 11.6 +/− 1.1 (3) 8.2 +/− 0.9 (3) 8.3 +/− 1.9(3) 7.8 +/− 0.8 (3) 7.7 +/− 1.2 (3) HNF4a 13.3 +/− 0.8 (3) 7.0 +/− 0.5(3) 6.1 +/− 0.0 (3) 7.7 +/− 1.4 (3) 6.9 +/− 1.9 (3) HNFβa 17.4 +/− 0.8(3) 7.8 +/− 1.1 (3) 5.0 +/− 1.3 (3) 5.5 +/− 0.5 (3) 4.5 +/− 1.0 (3)C/EBPa  7.9 +/− 0.4 (3) 5.8 +/− 0.3 (3) 6.1 +/− 0.4 (3) 6.0 +/− 0.0 (3)5.0 +/− 1.3 (3) AFP 21.2 +/− 2.4 (4) 2.0 +/− 0.7 (4) 0.8 +/− 0.7 (4) 0.8+/− 0.9 (4) 1.5 +/− 0.8 (4) TTR 16.7 +/− 1.8 (3) 2.8 +/− 1.2 (3) 0.6 +/−1.9 (3) 1.8 +/− 1.2 (3) 1.5 +/− 1.8 (3) CK19  8.8 +/− 0.6 (3) 5.0 +/−0.2 (3) 4.7 +/− 0.6 (3) 3.1 +/− 0.7 (3) 3.4 +/− 0.5 (3) Alb 19.4 +/− 1.4(4) 11.0 +/− 0.6 (4)  6.5 +/− 0.2 (4) 2.0 +/− 0.4 (4) 2.0 +/− 0.8 (4)CK18  3.9 +/− 1.1 (3) 1.3 +/− 0.2 (3) 0.1 +/− 1.1 (3) 1.0 +/− 0.5 (3)0.4 +/− 1.4 (3) AAT 22.5 +/− 1.1 (3) 9.4 +/− 2.1 (3) 3.1 +/− 0.6 (3) 2.4+/− 0.4 (3) 1.9 +/− 0.6 (3) TAT 22.9 +/− 0.9 (3) 16.2 +/− 0.8 (3)  10.6+/− 0.3 (3)  6.6 +/− 0.4 (3) 6.1 +/− 0.4 (3) G6P 23.7 +/− 1.1 (3) 19.3+/− 1.2 (3)  17.3 +/− 0.4 (3)  12.3 +/− 1.6 (3)  13.2 +/− 0.9 (3)  Cx3223.3 +/− 0.4 (3) 22.3 +/− 0.1 (3)  15.7 +/− 2.2 (3)  14.0 +/− 1.9 (3) 13.3 +/− 0.9 (3)  PEPCK 21.5 +/− 1.5 (3) 13.9 +/− 0.3 (3)  15.0 +/− 0.7(3)  16.1 +/− 1.1 (3)  14.9 +/− 0.7 (3)  CYP7A1 23.9 +/− 0.5 (3) 18.5+/− 1.5 (3)  19.3 +/− 1.9 (3)  20.8 +/− 2.5 (3)  22.5 +/− 1.7 (3) 

TABLE 1b Rat Clone R2Old rMAPC-1 d 0 d 6 d 12 d 16 d 20 Oct4  5.61 +/−0.9 (11)  10.6 +/− 2.09 (15) 12.94 +/− 0.8 (10) 11.36 +/− 0.9 (7) 10.615 +/− 1.19 (11)  Gsc  12.9 +/− 1.4 (10)  6.3 +/− 1.9 (17) 14.92 +/−1.2 (10) 13.35 +/− 1.08 (6)  15.84 +/−1.13 (7)  Tm4sf2 18.19 +/− 2.8(6)   2.6 +/− 0.9 (10)  6.68 +/− 0.35 (6) 7.97 +/− 1.19 (3) 7.27 +/− 0.8(6)  Mixl1 18.566 +/− 0.5 (5)  7.5 +/− 2 (7)  15.27 +/− 0.48 (5) 19.09+/− 0.4 (2)  16.96 +/− 2.1 (5)  eomes  8.66 +/− 0.98 (6)  3.7 +/− 1.4(9)  9.2 +/− 0.7 (5) 9.9 +/− 0.1 (2) 9.44 +/− 0.3 (4)  brachyuri ND NDND ND ND Lhx 11.17 +/− 0.4 (6)   3.6 +/− 1.09 (6) 9.96 +/− 0.7 (4) 12.69+/− 0.08    12.43 +/− 1.4 (4)  Sox7  6.7 +/− 2.1 (9)  6.16 +/− 1.47 (13)6.63 +/− 0.3 (7) 7.8 +/− 2.5 (5) 9.3 +/− 3 (8)  Sox17  2.5 +/− 1.35 (6)3.17 +/− 1.9 (8)  5.8 +/− 0.9 (4) 8.53 +/− 0.2 (2)  7.3 +/− 2 (4) Tmprss2 12.7 +/− 0.4 (4)  2.6 +/− 0.4 (7)  2.4 +/− 0.5 (5) 3.38 +/− 0.14(2) 2.77 +/− 0.17 (4) Foxa2  4.5 +/− 0.5 (4) 9.04 +/− 0.8 (4)  3.5 +/−0.2 (4) 4.46 +/− 0.9 4)  4.4 +/− 0.5 (6) Cxcr4 15.77 +/− 1.76 (9)   5.9+/− 1.5 (816) 9.36 +/− 0.9 (9) 9.8 +/− 1.04  9.3 +/− 0.6 (7) FlK1 19.9+/− 2 (5)  16.07 +/− 1.8 (7)  11.31 +/− 2.65 (8) 11.24 +/− 2.1 (5)  9.6+/− 1.6 (7) E-cadherin ND ND ND ND ND Hex1  8.9 +/− 0.4 (4) 3.83 +/− 0.4(8)  4.2 +/− 0.8 (6) 5.3 +/− 0.2 (4) 5.77 +/− 0.4 (8)  Prox1 9.167 +/−0.4 (5)   5.3 +/− 0.8 (8) 4.29 +/− 1.3 (8) 5.5 +/− 1.6 (4) 4.86 +/− 0.9(8)  Hnf1a 11.55 +/− 0.5 (6)  8.32 +/− 0.7 (8) 7.79 +/− 0.7 (7) 8.05 +/−0.4 (6)  7.78 +/− 0.5 (12) Hnf1b 6.86 +/− 1.1 (5) 6.68 +/− 0.9 (8)  6.37+/− 1.15 (5) 7.07 +/− 0.8 (4)  7.03 +/− 0.6 (12) Hnf6 17.02 +/− 2 (5)  9.19 +/− 1.4 (8) 5.27 +/− 1.2 (8) 4.87 +/− 0.2 (4)  5.22 +/− 0.8 (12)Cebpa  8.14 +/− 1.15 (5) 5.87 +/− 0.9 (8) 0.66 +/− 0.6 (6) 6.57 +/− 0.5(4)  5.72 +/− 0.3 (8)  Hnf4a 15.19 +/− 1.46 (5) 6.24 +/− 0.8 (7) 7.67+/− 1.7    6.018 +/− 1.3 (5)  6.96 +/− 1.5 (6)  AFP 21.07 +/− 2.2 (4) 5.17 +/− 2 (20)   0.01 +/− 0.76 (15) 0.4(neg) +− 9.02 (6) 0.2(neg) +/−0.76 (12)   TTR 15.5 +/− 0.9 (4)  2.89 +/− 1.3 (18)  0.32 +/− 0.63 (15)0.4 +/− 0.9 (6)  0.3 +/− 0.6 (12) CK19 7.42 +/− 0.2 (7)  3.8 +/− 0.5 (8) 3.1 +/− 0.5 (8) 3.727 +/− 0.5 (4)  1.87 +/− 0.15 (8) Alb 21.01 +/− 2.7(19) 13.94 +/− 1.2 (17)  8.9 +/− 1.7 (14) 6.24 +/− 1.55 (7) 1.14 +/− 1.8(20) CK18  0.8 +/− 2.15 (4) 0.8(neg) +/− 0.6 (9)     0.57 +/− 0.3 (7)0.85 +/− 0.4 (5)  0.7 +/− 0.2 (6) AAT 20.04 +/− 2.9 (10)  9.94 +/− 3.6(15)  2.6 +/− 0.5 (11) 0.76 +/− 1.15 (7)  1.3 +/− 1.03 (18) TAT 21.47+/− 2 (10)  13.07 +/− 2.3 (16)  9.3 +/− 1.7 (14) 7.4 +/− 0.8 (7)  4.3+/− 1.07 (30) G6P 23.06 +/− 1 (10)   16.5 +/− 1.6 (15) 16.07 +/− 1.8(13) 14.63 +/− 1.3 (10)  11.38 +/− 1.07 (25) Cx32 22.88 +/− 0.8 (10)19.7 +/− 3 (13)  15.89 +/− 2.6 (12) 13.62 +/− 0.8 (12)  12.8 +/− 0.9(25) PepcK 22.48 +/− 0.9 (4)  12.95 +/− 2 (4)   14.08 +/− 1 (4)   12.6+/− 1.2 (2)  11.29 +/− 1 (14)   Cyp7a1 23.21 +/− 2.17 (6) 19.75 +/− 2.2(5)  16.9 +/− 1.2 (5) 17.05 +/− 0.1 (3)  16.5 +/− 1.9 (15) Mrp2 19.84+/− 2.17 (8) 15.02 +/− 1.4 (11) 12.63 +/− 2 (11)  11.53 +/− 2.1 (8) 10.7 +/− 1.5 (18) Bsep 22.3 +/− 1.8 (8) 18.08 +/− 2.2 (10) 16.69 +/− 1.1(7)  15.35 +/− 0.5 (8)  13.2 +/− 1.4 (22) Arg1 14.79 +/− 0.8 (7)  9.28+/− 0.7 (9)  7.8 +/− 0.7 (10) 8.01 +/− 1.35 (8) 8.06 +/− 0.9 (22)

From these studies, the following conclusions were drawn:

a. Differentiation of MAPCs performed in the presence of the differentcytokines, in the order in which they are added to the medium, yielded afinal population of cells that expressed more mature hepatic transcriptscompared with MAPCs maintained in the absence of cytokines but in samemedium and at similar density. For some endodermal genes, such as AFPand TTR, expression increased earlier in the multi-step protocol. Theslighter upregulation of hepatic specific genes, even in the absence ofcytokines, suggested that cell-cell contact is important to initiate theendodermal differentiation process.

b. As a morphological heterogeneous cell population was obtained,expression of non-endodermal markers was evaluated. Also found weregenes expressed in endothelium (Flk1, PECAM) and a transientupregulation of smooth muscle transcripts (SM 22, calponin, SM actin).Expression of these non-hepatic transcripts was more similar between thetwo culture conditions.

c. During the first days of differentiation, Oct4 expression wasdownregulated. On day 6, there was a transient upregulation ofdefinitive endoderm specific transcripts, such as goosecoid and CXCR4.As in normal liver development, expression of these genes decreasedagain upon further maturation. Over time, slight down-regulation of theprimitive endodermal marker Sox 7 was observed.

d. A high concentration of dexamethasone and a low concentration ofinsulin resulted in enhanced expression of some mature markers (albumin,TAT).

Abbreviation of the markers and the positive control used for eachmarker is shown in Table 2.

TABLE 2 ABBREVI- POSITIVE ATION ABBREVIATION FOR CONTROL Alb AlbuminFetal liver E15 AAT Alpha1-antitrypsin Fetal liver E15 TAT Tyrosineaminotransferase Adult rat liver MRP2 Multidrug resistant protein 2Fetal liver E15 NTCP Na+/taurocholate-cotransporting Fetal liver E15polypeptide DPPIV Dipeptidyl-peptidase 4 Fetal liver E15 CX32 Connexin32 Fetal liver E15 GLY S Glycogen Synthase 1 Fetal liver E15 TRYPTTryptophan 2,3-dioxygenase Fetal liver E15 ARG1 Arginase type 1 Fetalliver E15 G6P Glucose-6-phosphatase Fetal liver E15 CYP7A1 Cholesterol 7α-hydroxylase Fetal liver E15 PK LIVER Liver specific pyruvate kinaseFetal liver E15 F VII Factor VII Fetal liver E15 TTR Transthyretin orpre-albumin Fetal liver E15 AFP Alpha-fetoprotein Fetal liver E15 CXCR4Chemokine receptor 4 Fetal liver E15 HNF1 Hepatocyte nuclear factor 1Fetal liver E15 HNF4a Hepatocyte nuclear factor 4 α Adult rat liver CK19Cytokeratin 19 Fetal liver E15 CEBPa CCAAT enhancer-binding Fetal liverE15 protein alpha FOXH1 forkhead box H1 = FAST Fetal liver E15 FGF8Fibroblast growth factor 8 Fetal liver E15 GSC Goosecoid Fetal liver E15SOX7 SRY-related HMG box proteins 7 Fetal liver E15 CL18 Cytokeratin 18Fetal liver E15 HEX Hematopoietically expressed Fetal liver E15 homeoboxHNF3B Hepatocyte nuclear factor 3B Fetal liver E15 C-MET HGF-receptorFetal liver E15 CK8 Cytokeratin 8 Fetal liver E15 GATA4 GATA family ofzinc finger- Fetal liver E15 containing transcription factors PROX1prospero-related homeobox 1 Fetal liver E15 PDX1 Pancreas DuodenumHomeobox-1 Fetal liver E15 SM22 Smooth muscle 22 Universal RNA CALPONCalponin Universal RNA S M ACT Smooth muscle actin Universal RNA FLK1VEGF-receptor-2 Universal RNA PECAM platelet/endothelial cell adhesionUniversal RNA molecule-1/CD31 NKX2.5 Early cardiac specific UniversalRNA transcription factor NK2 transcription factor related, locus 5 GATA6GATA family of zinc finger- Universal RNA containing transcriptionfactors BLBP Brain lipid binding protein Embryonic Brain EN1 Earlyforebrain marker Embryonic Brain NESTIN intermediate filament proteinEmbryonic Brain central nervous system progenitor marker OTX1Transcription factor specific for Embryonic Brain anterior neurectodermVIMENTIN cytoskeleton filament expressed Embryonic Brain in mesenchymalcells afterbirth SOX2 SRY-related HMG box proteins Embryonic Brain 2Early neuronal marker PAX6 Paired box gene 6; Developing Embryonic Braineye and pancreas and distinct domains of the CNS MHC-1 Majorhistocompatibility Universal RNA complex-1 OCT4 POU transcription factorfamily- Universal RNA Pluripotency marker

2.4 Immunohistochemistry

FIG. 5 A-F shows immunohistochemical staining for rAFP at differenttimepoints of differentiation of rMAPC. FIG. 5A shows rAFP stainingafter day 3 of differentiation. FIG. 5B shows rAFP staining after day 6of differentiation. FIG. 5C shows rAFP staining after day 12 ofdifferentiation. FIG. 5D shows rAFP staining after day 16 ofdifferentiation. FIG. 5E shows rAFP staining after day 20 ofdifferentiation. FIG. 5F shows rAFP staining after day 20 in the isotypecontrol.

FIG. 6 A-F shows immunohistochemical staining for rALB at differenttimepoints of differentiation of rMAPC. FIG. 6A shows rALB staining ofundifferentiated rMAPC at day 0. FIG. 6B shows rALB staining after day12 of differentiation. FIG. 6C shows rALB staining after day 16 ofdifferentiation. FIG. 6D shows rALB staining after day 20 ofdifferentiation. FIG. 6E shows rALB staining in rat hepatocytes. FIG. 6Fshows rALB staining after day 20 in the isotype control.

FIG. 7 shows immunohistochemical staining for CK18 at differenttimepoints of differentiation of rMAPC-1. The top three lines are,respectively, CK18 staining in undifferentiated rMAPC-1 at day 0; CK18staining after day 20 of differentation; and CK18 staining in mature rathepatocytes. The lower three lines show the isotype controls used at day0, after day 20 and in mature rat hepatocytes, respectively, in theisotype control.

2.6 Functional Assays

a. Secretion of Albumin in the Medium

Using rat albumin specific ELISA, increasing amounts of albumin could bedetected in the conditioned medium of differentiating rat MAPCs afterdays 12, 14, 16, 18 and 20 (FIG. 8). No albumin was detected in themedium of cells grown in control medium without cytokines. Decreasingthe concentration of dexamethasone resulted in reduced secretion ofalbumin.

b. Glycogen Storage

As early as day 14 of differentiation, a significant amount of cellsstained positive for PAS staining. FIG. 9 shows the concentration ofglycogen (μg/mg protein) for rMAPC (n=6-10).

c. LDL-Uptake

LDL-uptake is a characteristic of both endothelial cells andhepatocytes. The difference between these two cells was determined bythe faster up-take of LDL by hepatocytes (2 hours) compared toendothelial cells (4 hours) (data not shown).

d. Lectin Staining

A subpopulation of cells stained positive for Lectin, which is anendothelial marker, at day 18 of differentiation, as was suggested byqRT-PCR results (data not shown). During liver embryology, contact ofhepatoblasts with endothelial cells is necessary for their furthermaturation. Thus, the inventors hypothesized that a heterogeneous cellpopulation, including endothelial cells, may have beneficial effects onthe maturation of adjacent hepatic-like cells.

e. Conjugation of Bilirubin

In vivo, bilirubin is a degradation product of the heme moiety ofhemoglobin and heme proteins such as cytochromes, muscle myoglobin andcatalase. Because unconjugated bilirubin is unsoluble, it is transportedas an albumin-bound complex (90%). Intrahepatic transport ofunconjugated bilirubin to the endoplasmatic reticulum occurs by bindingto ligandin (glutathione S-transferase B) and/or protein Z. Efficientexcretion of bilirubin across the bile canaliculi requires theconversion of unconjugated bilirubin to polar conjugates byesterification of one or two propionic acid side chains of the bilirubinmolecule, with a sugar, mostly glucuronic acid, to form water-solublemono or diglucuronated bilirubin, respectively. This is done by uridinediphosphate-glucuronyltransferase 1A1. Bilirubin glucuronides areexcreted into bile canaliculi by the multidrug resistance associatedprotein 2 (MRP2), a member of the ATP-cassette transporter family. Innormal human bile, 80% of the conjugated bilirubin exists as bilirubindiglucuronide, and only a minor portion exists in the monoglucuronidestate. In rats, in contrast, monoconjugated bilirubin is the majorfraction.

FIG. 10 shows an increasing formation of monoconjugated bilirubin after4, 8, 12, 22 and 33 hours in mature rat hepatocytes (FIG. 10A) and indifferentiated rMAPC after day 20 (FIG. 10B).

In conclusion, a 21 day differentiation protocol resulted in aheterogeneous cell population containing hepatic-like cells, but alsoepithelial, endothelial and smooth muscle cells. Although only a limitedamount of cells were mature hepatocytes, they secreted a significantamount of albumin, took up LDL, conjugated unconjugated bilirubintowards monoglucuronated bilirubin, showed signs of glycogen storage,and, as shown using electron microscopy, their morphology was consistentwith biliary and hepatic cells.

Example 2

I. Methodology

The human embryonic stem cell (hESC) line, H9, and induced pluripotentstem cell (hIPSC) were differentiated according to the standardprotocol. At different time points (prior to cytokine exposure, at thethree time points following exposure to the three other sets ofcytokines as described herein, and the final time point representing theendpoint cell type), samples were obtained for RT-qPCR analysis of PSgenes, ME/DE genes, hepatoblasts and definitive hepatocyte genes. Inaddition, cultures were evaluated for secretion of albumin, conversionof NH₃ to urea, glycogen storage, metabolization of bilirubin, GSTactivity, and expression of coagulation factors Factor V, Factor VII,Protein C, gamma-glutamylcarboxylase (GGCX) and Vitamin K epoxidereductase (VKOR).

II. Results

The RT-qPCR results are shown in FIG. 11 A-J. The RT-qPCr results alsoare shown in Table 3a-b. Table 3a and 3b provide values for expressionof genes in the human ESC line H9 and the hiPS cells, respectively, asdescribed in the methodology.

TABLE 3a Gene Expression of Human ESC line H9 hESC (H9) d 0 d 6 d 10 d14 d 20 Oct4 .−0.3 +/− 1.0 (8)  .−0.2 +/− 1.1 (12)  3.3 +/− 1.4 (8)  5.3+/− 2.0 (10) 6.4 +/− 2.1 (8) Mixl1  8.6 +/− 1.3 (3) 3.9 +/− 1.0 (6) 9.5+/− 0.4 (4) 9.9 +/− 1.6 (4) 9.7 +/− 2.5 (3) Eomes 10.7 +/− 2.7 (4) 2.5+/− 1.4 (6) 7.0 +/− 1.4 (6) 6.7 +/− 1.7 (4) 15.5 +/− 2.5 (4)  Brachyury10.4 +/− 2.5 (5) 5.9 +/− 1.3 (7) 9.7 +/− 1.7 (3) 12.3 +/− 1.0 (7)  13.4+/− 0.8 (5)  Tm4sf2  8.0 +/− 0.6 (3) 7.0 +/− 0.7 (3) 8.0 +/− 0.6 (3) 9.0+/− 0.3 (3) 6.6 +/− 0.5 (3) Sox7 13.1 +/− 0.5 (3) 13.1 +/− 0.9 (3)  9.7+/− 1.3 (3) 11.4 +/− 0.4 (3)  10.5 +/− 0.4 (3)  Gsc 13.2 +/− 3.9 (4) 6.6+/− 1.8 (6) 12.8 +/− 2.4 (6)  11.8 +/− 2.7 (4)  12.7 +/− 1.6 (4)  Sox17 7.9 +/− 2.8 (4) 4.5 +/− 2.0 (7) 4.6 +/− 1.4 (5) 6.5 +/− 2.0 (5) 7.7 +/−1.4 (4) Foxa2 12.4 +/− 3.1 (6)  9.5 +/− 2.4 (10) 6.7 +/− 1.7 (6) 9.3 +/−3.2 (8) 10.2 +/− 1.8 (6)  Cxcr4  6.3 +/− 2.5 (4) 3.9 +/− 1.1 (7) 6.4 +/−1.4 (5) 4.7 +/− 1.5 (5) 6.5 +/− 1.0 (4) Flk1  7.3 +/− 0.2 (3) 6.9 +/−0.2 (3) 5.6 +/− 0.5 (3) 6.5 +/− 0.7 (3) 3.7 +/− 0.1 (3) E-cadherin  7.7+/− 2.1 (7) 10.2 +/− 3.0 (7)  9.6 +/− 2.7 (6) 9.6 +/− 3.5 (7) 7.7 +/−2.8 (7) Hex1 + + + + + Prox1 10.0 +/− 0.5 (3) 8.4 +/− 0.2 (3) 6.2 +/−0.1 (3) 5.3 +/− 1.6 (3) 5.8 +/− 0.5 (3) HNF1a 14.4 +/− 0.2 (3) 13.6 +/−1.3 (3)  6.8 +/− 1.5 (3) 8.7 +/− 2.0 (3) 7.9 +/− 0.9 (3) HNF4a 11.2 +/−3.2 (3) 7.1 +/− 1.6 (3) 4.1 +/− 1.8 (4) 9.5 +/− 4.9 (3) 2.7 +/− 0.5 (3)HNF6a 21.6 +/− 0.2 (3) 21.0 +/− 0.9 (3)  21.7 +/− 0.7 (3)  21.2 +/− 0.4(3)  20.2 +/− 0.4 (3)  C/EBPa 10.7 +/− 0.1 (3) 11.7 +/− 1.2 (3)  5.8 +/−1.7 (3) 8.9 +/− 1.9 (3) 6.6 +/− 0.6 (3) AFP 20.9 +/− 1.3 (3) 19.5 +/−2.1 (3)  14.5 +/− 1.6 (4)  14.1 +/− 0.3 (3)  11.8 +/− 0.3 (3) TTR + + + + + CK19  2.3 +/− 0.9 (5) 1.7 +/− 0.8 (7) .−1.1 +/− 0.4 (4) 0.5 +/− 1.1 (7) 1.2 +/− 0.6 (5) Alb 21.4 +/− 2.4 (8) 18.8 +/− 4.4 (9) 9.8 +/− 1.5 (7) 6.9 +/− 2.4 (9) .−2.0 +/− 2.0 (8)  CK18  1.6 +/− 0.2 (3)1.5 +/− 0.6 (3) 0.5 +/− 0.5 (3) 0.8 +/− 0.4 (3) .−0.1 +/− 0.8 (1)  AAT18.0 +/− 2.7 (6) 18.0 +/− 4.0 (8)  10.6 +/− 6.0 (4)  10.2 +/− 5.9 (8) 4.8 +/− 3.8 (6) TAT + + + + + G6P 21.7 +/− 0.7 (3) 15.2 +/− 3.9 (3) 15.2 +/− 2.7 (3)  19.2 +/− 2.4 (3)  16.8 +/− 1.5 (3)  Cx32 18.6 +/− 1.7(3) 19.8 +/− 1.8 (3)  8.7 +/− 2.8 (3) 12.5 +/− 3.7 (3)  10.0 +/− 1.9(3)  PEPCK + 4.6 +/− 0.8 (3) 3.7 +/− 0.3 (2) 3.9 +/− 0.5 (3) 6.4 +/− 2.0(3) CYP1A2 19.7 +/− 1.5 (3) 17.8 +/− 3.6 (3)  15.6 +/− 3.9 (2)  22.0 +/−1.2 (3)  20.4 +/− 0.1 (3)  CYP3A4 21.7 +/− 0.7 (3) 21.7 +/− 0.3 (3) 21.8 +/− 0.8 (4)  22.6 +/− 0.4 (3)  20.7 +/− 0.9 (3) 

TABLE 3b Gene Expression of hiPS cells hiPS d 0 d 6 d 10 d 14 d 20 Klf4Tg 22.1 (1)  20.9 (1) 21.5 (1) 19.7 (1) 19.3 (1) Sox2 Tg 19.6 +/− 3.6(2)  18.4 +/− 3.5 (2)  19.1 +/− 2.8 (2) 19.7 (1) 19.3 (1) Oct4 Tg 21.5+/− 0.9 (2)  20.5 +/− 0.6 (2)  21.3 +/− 0.2 (2) 19.4 (1) 19.3 (1) Rex8.3 (1) 13.5 (1) 12.7 (1) 18.4 (1) 17.0 (1) Nanog 4.8 +/− 0.5 (2) 8.4+/− 0.0 (2) 16.7 +/− 0.9 (2) 18.1 (1) 17.6 (1) Sox2 endo 5.6 +/− 0.4 (2)9.0 +/− 0.2 (2) 16.8 +/− 0.5 (2) 15.7 (1) 16.9 (1) Oct4 endo 8.8 +/− 0.7(4) 13.2 +/− 1.4 (4)  19.5 +/− 1.9 (4) 17.2 +/− 3.3 (3)  16.3 +/− 2.2(3)  Mixl1 4.8 +/− 0.7 (3) 4.0 +/− 1.5 (3) 10.4 +/− 1.9 (3) 9.0 +/− 2.0(3) 9.4 +/− 1.4 (3) Eomes 4.4 +/− 1.0 (3) 3.8 +/− 1.5 (3)  9.0 +/− 1.8(3) 8.6 +/− 2.8 (3) 10.5 +/− 0.6 (3)  Brachyury 5.6 (1)  6.1 (1)  7.8(1)  8.5 (1)  7.5 (1) Tm4sf2 7.8 +/− 0.7 (3) 7.0 +/− 0.7 (3)  9.9 +/−1.4 (3) 10.3 +/− 1.5 (3)  6.9 +/− 0.4 (3) Sox17 6.4 +/− 1.2 (3) 5.5 +/−1.9 (3)  7.9 +/− 2.5 (3) 9.0 +/− 1.4 (3) 8.3 +/− 1.1 (3) Foxa2 9.9 +/−2.9 (3) 10.3 +/− 1.3 (3)  10.6 +/− 1.5 (3) 12.0 +/− 1.6 (3)  11.3 +/−1.7 (3)  Cxcr4 3.5 +/− 3.1 (3) 3.2 +/− 2.2 (3)  7.4 +/− 2.5 (3) 6.7 +/−1.6 (3) 7.2 +/− 1.3 (3) Flk1 8.2 +/− 2.2 (3) 10.5 +/− 2.4 (3)  13.0 +/−6.4 (3) 14.4 +/− 4.6 (3)  10.1 +/− 3.3 (3)  E-cadherin 4.5 (1)  4.9 (1) 3.4 (1)  4.6 (1)  2.5 (1) HNF4a 7.1 +/− 2.0 (3) 7.4 +/− 0.7 (3)  6.4+/− 1.9 (2) 8.3 +/− 0.3 (3) 6.4 +/− 0.2 (3) AFP 19.9 +/− 0.7 (3)  17.9+/− 1.3 (3)  17.3 +/− 0.8 (3) 18.3 +/− 1.5 (3)  13.8 +/− 1.8 (3) TTR + + + + + CK19 1.7 +/− 1.3 (3) 3.2 +/− 3.0 (3)  2.9 +/− 3.4 (3) 3.2+/− 2.5 (3) 1.4 +/− 2.6 (3) Alb 15.9 +/− 2.8 (3)  19.2 +/− 1.7 (3)  11.1+/− 4.7 (3) 6.5 +/− 2.0 (3) 0.3 +/− 3.2 (3) CK18 0.7 +/− 0.8 (3) 1.9 +/−1.0 (3)  3.7 +/− 2.5 (3) 3.2 +/− 1.8 (3) 0.1 +/− 0.7 (3) AAT 16.9 +/−2.8 (3)  19.2 +/− 1.7 (3)  18.4 +/− 1.6 (3) 14.8 +/− 0.9 (3)  9.2 +/−3.5 (3) TAT + + + + + G6P + + + + + Cx32 16.7 +/− 0.7 (3)  17.7 +/− 1.0(3)  13.7 +/− 2.0 (3) 15.2 +/− 0.3 (3)  11.3 +/− 0.7 (3)  PEPCK 5.8 +/−1.4 (3) 6.4 +/− 0.5 (3)  7.3 +/− 0.5 (3) 6.1 +/− 1.1 (3) 6.2 +/− 0.5 (3)CYP7A1 20.0 +/− 0.9 (2)  17.2 +/− 0.7 (2)  17.7 +/− 0.1 (2) 17.6 +/− 1.2(2)  16.6 +/− 1.2 (2) 

FIG. 11A shows the expression of primitive streak (PS) genes Oct 4,Mix/1, Eomes, brachyury and Gsc at day 0 and after days 2, 4, 6 and 10of differentiation. FIG. 11B shows the expression ofmesoendoderm/definitive endoderm (ME/DE) genes Sox 17, Foxa2, CXCR4,E-cadherin and Sox7 at day 0 and after days 2, 4, 6 10 ofdifferentiation. FIG. 11C shows the expression of hepatoblast genes AFP,TTR, CK19, A1b and CK18 at day 0 and after days 6, 10, 14 and 20 ofdifferentiation. FIG. 11D shows the expression of definitive hepatocytegenes Alb, AAT, TAT, G6P, Cx32, PEPCK, CYP1A2 and CYP3A4 at day 0 andafter days 6, 10, 14 and 20 of differentiation. FIG. 11E shows theconcentration of albumin secreted by the H9 cell cultures. FIG. 11Fshows the spontaneous production of urea and the conversion of HN₄HCO₃(1 mM) to urea of the H9 cells. FIG. 11G shows glycogen storage of theH9 cells at day 0 and after days 6, 10, 14 and 20 of differentiation.FIG. 11H shows the metabolization of different concentrations ofunconjugated bilirubin at day 0 and after days 6, 10, 14 and 20 ofdifferentiation. FIG. 11I shows the total GST activity at day 0 andafter days 6, 10, 14 and 20 of differentiation. FIG. 11J shows theexpression of coagulation factors Factor V, Factor VII, Protein C, GGCXand VKOR at day 0 and after days 6, 10, 14 and 20 of differentiation.FIG. 12 shows the concentration of albumin secreted by hiPS cells.

While the invention has been particularly shown and described withreference to preferred embodiments thereof, it will be understood bythose skilled in the art that various alterations in form and detail maybe made therein without departing from the spirit and scope of theinvention, as defined by the appended claims.

All references cited herein are incorporated by reference for theteachings referred to in citing these references.

What is claimed is:
 1. A method for inducing cells to differentiate intocells that express a hepatocyte phenotype, said method comprising thesteps of: (a) culturing either mouse or human embryonic stem (ES) cells,mouse or human induced pluripotent stem (iPS) cells or mouse, rat, orhuman multipotent adult progenitor cells characterized in that themultipotent adult progenitor cells can differentiate into at least onecell type of each of the endodermal, ectodermal, and mesodermalembryonic lineages but are not embryonic germ cells, embryonic stemcells, or germ cells, with about 5 ng/ml to about 500 ng/ml Wnt3a andabout 10 ng/ml to about 1,000 ng/ml ActivinA, to obtain cells thatexpress a definitive endoderm phenotype, (b) then culturing the cellsproduced in step (a) with about 1 ng/ml to about 100 ng/ml bFGF andabout 5 ng/ml to about 5 ng/ml to about 50 ng/ml BMP4, to obtain cellsthat express a liver committed endodermal phenotype, (c) then culturingthe cells produced in step (b) with about 5 ng/ml to about 500 ng/mlaFGF, about 1 ng/ml to about 100 ng/ml FGF4 and about 2.5 ng/ml to about250 ng/ml FGF8b, to obtain cells that express a hepatoblast phenotype,and (d) then culturing the cells produced in step (c) with about 2 ng/mlto about 200 ng/ml HGF and about 10 ng/ml to about 1,000 ng/mlFollistatin, to obtain cells that express a hepatocyte phenotype.
 2. Themethod of claim 1, wherein the cells are cultured in step (a) with about50 ng/ml Wnt3a and about 100 ng/ml ActivinA.
 3. The method of claim 1,wherein the cells are cultured in step (b) with about 10 ng/ml bFGF andabout 50 ng/ml BMP4.
 4. The method of claim 1, wherein the cells arecultured in step (c) with about 50 ng/ml aFGF, about 10 ng/ml FGF4andabout 25 ng/ml FGF8b.
 5. The method of claim 1, wherein the cells arecultured in step (d) with about 20 ng/ml HGF and about 100 ng/mlFollistatin.
 6. The method of claim 1, wherein the cells are cultured atone or more steps in a medium containing a serum concentration rangingfrom 0% to about 2%.
 7. The method of claim 1, wherein the cells arecultured at one or more steps in a medium containing about 10⁻⁵ M toabout 10⁻¹⁰ M dexamethasone.
 8. The method of claim 1, wherein the cellsare cultured at one or more steps for at least four days.
 9. The methodof claim 1, wherein the cells in step (a) are cultured for about sixdays, the cells in step (b) are cultured for about four days, the cellsin step (c) are cultured for about four days, and the cells in step (d)are cultured for about seven days.
 10. The method of any of claims 1-9,wherein the multipotent adult progenitor cells are derived from bonemarrow.